FluoroFinder

OMIP-083

A 21-marker 18-color flow cytometry panel for in-depth phenotyping of human peripheral monocytes

https://doi.org/10.1002/cyto.a.24545

Kathryn E. Hally, Laura Ferrer-Font, Katherine R. Pilkington, Peter D. Larsen

Description: This 21-marker, 18-color panel was developed to accurately delineate monocyte subsets by manual gating and, once successfully gated, to characterize monocyte function in-depth. This panel was optimized on human peripheral blood mononuclear cells (PBMCs) density-separated from whole blood drawn into Cyto-Chex blood collection tubes (BCTs; Streck, La Vista, NE) from healthy adults.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: Aurora 3L 16V-14B-8R

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD64	Human	10.1	Brilliant Violet 605	Fc receptor expression
CD16	Human	3G8	Brilliant Violet 421	ID monocyte subsets, Fc receptor expression
Slan	Human	DD-1	VioBlue	ID monocyte subsets
HLA DR	Human	G46-6	Brilliant Violet 480	Antigen presentation
CD36	Human	AC106	VioGreen	Scavenger receptor expression
CD14	Human	M5E2	Brilliant Violet 570	ID monocyte subsets
CD11b	Human	ICRF44	Brilliant Violet 650	Fc receptor expression
PD-1	Human	EH12.2H7	Brilliant Violet 750	Examine the PD-1 pathway
CD184	Human	12G5	Brilliant Violet 785
CD3	Human	SK7	FITC	Exclude pan-T cells
CD123	Human	6H6	FITC	Exclude basophils and pDCs
CD19	Human	SJ25C1	FITC	Exclude B cells
CD56	Human	HCD56	FITC	Exclude NK cells
CD45	Human	HI30	Alexa Fluor 532	ID leukocytes
TLR2	Human	11G7	PE	ID monocytes, pattern recognition receptor expression
CD66b	Human	G10F5	PE-Dazzle 594	Exclude neutrophils and eosinophils
CD11c	Human	3.9	PE-Cy5	Adhesion receptor expression
CX3CR1	Human	2A9-1	PerCP-Cy5.5	Cytokine receptor expression
CD86	Human	BU63	PE-Cy7
CD42b	Human	HIP1	APC	Heterotypic aggregation
CD192	Human	K036C2	APC-Fire 750	ID monocyte subsets, cytokine receptor expression

OMIP-082

A 25-color phenotyping to define human innate lymphoid cells, natural killer cells, mucosal-associated invariant T cells, and γδ T cells from freshly isolated human intestinal tissue

https://doi.org/10.1002/cyto.a.24529

Chloe M. Doyle, Nicole L. Fewings, Grahame Ctercteko, Scott N. Byrne, Andrew N. Harman, Kirstie M. Bertram

Description: Extensive phenotyping of human ILCs, MAIT, NK and γδ T cells

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: FACS Symphony

Panel details

Marker	Species	Clone	Fluorophore	Purpose	Co-expressions groups	Antigen Density
CD336		p44-8	Brilliant Blue 515

OMIP-081

A new 21-monoclonal antibody 10-color panel for diagnostic polychromatic immunophenotyping

TBD

Erik H. L. P. G. Huys,Willemijn Hobo,Frank W. M. B. Preijers

Description: The 10-color panel consisting of 21 monoclonal antibodies (mAbs) is developed as a one-tube panel to detect leukemia and lymphoma cells in all hematopoietic cell lineages. In particular, this tube is mentioned for a fast screening to identify aberrant cells in samples suspected for malignant cell localization and to enable comprehensive immunophenotyping of samples with low cell counts.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: Beckman Coulter Navios

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD8	Human	B9.11	Pacific Blue	Cytotoxic T cells, NK-cell subset
CD4	Human	SFCI12T4D11	PE-Cy7	Helper T cells, Monocyte lineage
CD3	Human	UCHT1	APC-Alexa 750	Mature T cells
CD45	Human	J33	Krome Orange	WBC subpopulations
CD2	Human		APC-Alexa 700	T-cell lineage, NK-cell subset
CD7	Human	8H8.1	FITC	T-cell lineage, T-cell activation, NK-cell subsets
CD19	Human		ECD	B-cell lineage
Ig Kappa Light Chain	Human		APC	B-cell subset, B-cell lineage clonality
Ig Lambda Light Chain	Human		PE	B-cell subset, B-cell lineage clonality
CD138	Human	B-B4	FITC	Plasma cells
CD36	Human	FA6.152	APC-Alexa 700	Monocyte lineage, erythroid lineage, megakaryocytes
CD10	Human	ALB1	APC-Alexa 700	B-cell precursors, mature neutrophils
CD117	Human	104D2D1	APC	Myeloid precursors
CD34	Human	581	PE-Cy7	Progenitor cells
CD15	Human	80H5	Pacific Blue	Myeloid lineage
CD33	Human	D3HL60.251	PE-Cy5.5	Monocyte lineage, myeloid lineage
CD20	Human	B9E9	Pacific Blue	Mature B cells
CD56	Human	NCAM16.2	PE	NK cells, plasma cell subset
CD16	Human	B73.1	PE	T-cell subsets, NK cells, monocyte differentiation, eosinophil exclusion, myeloid lineage
CD5	Human	BL1a	PE-Cy5.5	T-cell lineage, B-cell subset
CD14		RMO52	ECD	Mature monocytes

OMIP-080

29-Color flow cytometry panel for comprehensive evaluation of NK and T cells reconstitution after hematopoietic stem cells transplantation

TBD

Sarka Vanikova,Abhishek Koladiya,Jan Musil

Description: This 29-color panel was developed and optimized for the monitoring of NK cell and T cell reconstitution in peripheral blood of patients after HSCT.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD62L	Human	DREG-56	Brilliant Ultraviolet 395	T cell differentiation
Live/Dead Fix Blue	All Species		Live/Dead Fix Blue	Viability
CD69	Human	FN50	Brilliant Ultraviolet 496	NK and T cell activation marker
CCR6	Human	11A9	Brilliant Ultraviolet 563	Chemokine receptor; Th subset identification
CD27	Human	M-T271	Brilliant Ultraviolet 615	T cell differentiation
PD-1	Human	EH12.1	Brilliant Ultraviolet 661	T cell inhibitory receptor
CD25	Human	2A3	Brilliant Ultraviolet 737	T cell activation; Treg identification
CD8	Human	RPA-T8	Brilliant Ultraviolet 805	CD8 T cell and NKT-like cell lineage marker
KLRC1	Human	131411	Brilliant Violet 421	NK and NKT-like cell inhibitory receptor
CD45RA	Human	HI100	Pacific Blue	T cell differentiation
TIM-3	Human	7D3	Brilliant Violet 480	T cell inhibitory receptor
CD4	Human	RPA-T4	Brilliant Violet 570	CD4 T cell lineage marker
CD57	Human	QA17A04	Brilliant Violet 605	T cell and NK cell differentiation
CD95	Human	DX2	Brilliant Violet 650	T cell activation and differentiation
TCR gamma/delta	Human	11F2	Brilliant Violet 711	γδ T cells
CD226	Human	DX11	Brilliant Violet 750	T cell and NK cell activating receptor
CD31	Human	WM59	Brilliant Violet 786	Adhesion molecule; identification of RTE
CCR10	Human	1B5	Brilliant Blue 515	Chemokine receptor; Th subset identification
CD194	Human	[1G1]	Brilliant Blue 700	Chemokine receptor; Th subset identification
TIGIT	Human	741182	Brilliant Blue 755-P	T cell and NK cell inhibitory receptor
CD314	Human	1D11	Brilliant Blue 790-P	NK cell activating receptor
KLRC2	Human	134591	PE	NK cell activating receptor
CD56	Human	NCAM16.2	PE-CF594	NK cell and NKT-like cell lineage marker
CD183	Human	1C6/CXCR3	PE-Cy5	Chemokine receptor; Th subset identification
FoxP3	Human	PCH101	PE-Cy5.5	Master transcription factor for Tregs
CD39	Human		PE-Cy7	Treg activation marker
CD3	Human	UCHT1	Alexa Fluor 647	T cell and NKT-like cell lineage marker
CD16	Human	3G8	Alexa Fluor 700	NK cell differentiation
Perforin	Human	B-D48	APC-Fire 750	Cytolytic function

OMIP-079

Cell cycle of CD4+ and CD8+ naïve/memory T cell subsets, and of Treg cells from mouse spleen

TBD

Ambra Natalini,Sonia Simonetti,Gabriele Favaretto,Giovanna Peruzzi,Fabrizio Antonangeli,Angela Santoni,Miguel Muñoz-Ruiz,Adrian Hayday,Francesca Di Rosa

Description: A multicolor flow cytometry panel was designed and optimized to define the following nine mouse T cell subsets: Treg (CD3+ CD4+ CD8− FoxP3+), CD4+ T naïve (CD3+ CD4+ CD8−FoxP3− CD44int/low CD62L+), CD4+ T central memory (CD3+ CD4+ CD8− FoxP3− CD44high CD62L+), CD4+ T effector memory (CD3+ CD4+ CD8− FoxP3− CD44high CD62L−), CD4+ T EMRA (CD3+ CD4+ CD8− FoxP3− CD44int/low CD62L−), CD8+ T naïve (CD3+ CD8+ CD4− CD44int/low CD62L+), CD8+ T central memory (CD3+ CD8+ CD4− CD44high CD62L+), CD8+ T effector memory (CD3+ CD8+ CD4− CD44high CD62L−), and CD8+ T EMRA (CD3+ CD8+ CD4− CD44int/low CD62L−). In each T cell subset, a dual staining for Ki-67 expression and DNA content was employed to distinguish the following cell cycle phases: G0 (Ki67−, with 2n DNA), G1 (Ki67+, with 2n DNA), and S-G2/M (Ki67+, with 2n < DNA ≤ 4n). This panel was established for the analysis of mouse (C57BL/6J) spleen.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD3	Mouse	145-2C11	FITC	Pan T cell marker
CD44	Mouse	IM7	APC	NaÏve/Memory subset identification
Ki-67	Mouse	SolA15	Alexa Fluor 700	Quiescence/cell cycle
Hoechst (33342)	All Species		Hoechst (33342)	DNA content/cell cycle
FoxP3	Mouse	FJK-16s	PE	Treg identification
CD4	Mouse	RM4-5	PE-CF594	Helper T cell identification
CD62L	Mouse	MEL-14	PE-Cy7	NaÏve/Memory subset identification
CD8	Mouse	53-6.7	Brilliant Violet 785	Cytotoxic T cell identification
CD16/CD32	Mouse	2.4G2	Purified
eFluor 780 Fix Viability	All Species		eFluor 780 Fix Viability	Live/Dead cell discrimination

OMIP-078

A 31-parameter panel for comprehensive immunophenotyping of multiple immune cells in human peripheral blood mononuclear cells

Cytometry PART A, Volume 99, Issue 9, 893-898 (2021)

Takuto Nogimori, Yuko Sugawara, Masaya Higashiguchi, Hirotomo Murakami, Hirofumi Akita, Shokichi Takahama, Satoshi Tanaka, Takuya Yamamoto

Description: This 31-parameter panel was developed for simultaneously measuring multiple immune cell populations including T cells, B cells, natural killer cells, dendritic cells, monocytes, and hematopoietic progenitor cells in human peripheral blood mononuclear cells. This panel enables the capture of individual immune dynamics and assessments of single-cell changes in the immune system that are associated with aging and diseases. This panel includes markers to separate the differentiation status of each cell population and might be applicable to studies of infectious and autoimmune diseases, as patient samples are usually limited in volume and require an analysis system that provides a relatively large amount of information.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
FVS700	All Species		FVS700	Viability
CD57	Human	NK-1	FITC
CD127	Human	HIL-7R-M21	Brilliant Blue 630-P2
CD38	Human	HIT2	Brilliant Blue 660-P2
CD34	Human	8G12	Brilliant Blue 700
CD1c	Human	F10/21A3	Brilliant Blue 755-P
IgM	Human (Negative)	G20-127	Brilliant Blue 790-P
CD133	Human	W6B3C1	PE
CD11c	Human		PE-CF594
CD56	Human	B159	PE-Cy5
CD4	Human	SK3	PE-Cy5.5
CD27	Human	M-T271	PE-Cy7
IgD	Human	IA6-2	APC
CD3	Human	SP34-2	APC-Cy7
CD303	Human	V24-785	Brilliant Violet 421
CD123	Human	7G3	Brilliant Violet 480
CD45RO	Human	UCHL1	Brilliant Violet 570
CD138	Human	MI15	Brilliant Violet 605
CD16	Human	3G8	Brilliant Violet 650
CD19	Human	SJ25C1	Brilliant Violet 711
CD24	Human	ML5	Brilliant Violet 750
PD-1	Human	EH12.1	Brilliant Violet 786
CD11b	Human	ICRF44	Brilliant Ultraviolet 395
CD45	Human	HI30	Brilliant Ultraviolet 496
CD8	Human	RPA-T8	Brilliant Ultraviolet 563
CD141	Human	1A4	Brilliant Ultraviolet 615
HLA-DR	Human	G46-6	Brilliant Ultraviolet 661
IgG	Human	G18-145	Brilliant Ultraviolet 737
CD14	Human	M5E2	Brilliant Ultraviolet 805

OMIP-077

Definition of all principal human leukocyte populations using a broadly applicable 14-color panel

TBD

Maximilian Boesch, Martina Sykora, Silvia Gasteiger, Florent Baty, Martin H. Brutsche, Sieghart Sopper

Description: The present optimized multicolor immunofluorescence panel (OMIP) provides for the detection and in-depth analysis of all major leukocyte subsets in human whole blood (WB) using a single flow cytometry (FCM) panel with just 14 colors. The panel offers an easy, fast and reproducible way of comprehensive immune cell profiling for various purposes including translational research and companion immune monitoring of clinical trials.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD14	Human	M?P9 (also known as M?P-9)	Brilliant Violet 711	Monocytes (c), intermediate monocytes, monocytes (nc)
CD15	Human	HI98	PerCP-Cy5.5	Eosinophils
CD16	Human	eBioCB16 (CB16)	APC-eFluor 780	Neutrophils, intermediate monocytes, monocytes (nc)
CD19	Human	HIB19	APC-R700	B cells, plasma cells
CD34	Human	8G12 (also known as HPCA2)	FITC	Progenitor cells
CD38	Human	HIT2	Brilliant Violet 421	Plasma cells, basophils
CD45	Human	HI30	Brilliant Violet 480	Pan-leukocytes
CD141	Human	AD5-14H12	APC	CD141+ mDCs
CD193	Human	5E8 (also known as 5E8-G9-B4)	PE-CF594	Eosinophils, basophils
HLA-DR	Human	G46-6	Brilliant Violet 786	Neutrophils, monocytes (c), intermediate monocytes, monocytes (nc), B cells, plasma cells, CD1c+ mDCs, CD141+ mDCs
CD1c	Human	AD5-8E7	PE	CD1c+ mDCs, MZB cells
CD3	Human	OKT3	Brilliant Violet 605	T cells
CD56	Human	NCAM16.2 (also known as NCAM 16)	PE-Cy7	NK cells
CD123	Human	7G3	Brilliant Violet 650	Basophils, pDCs

OMIP-076

High-dimensional immunophenotyping of murine T-cell, B-cell, and antibody secreting cell subsets

Cytometry PART A, Volume 99, Issue 9, 888-892 (2021)

Kyle T. Mincham, Jacob D. Young, Deborah H. Strickland

Description: This 19-parameter, 18-color flow cytometry panel was designed and optimized to enable the comprehensive and simultaneous immunophenotyping of distinct T-cell, B-cell, and antibody secreting cell (ASC) subsets within murine tissues.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD273	Mouse	TY25	Brilliant Ultraviolet 395	Memory B-cells
IgD	Mouse	AMS 9.1	Brilliant Ultraviolet 496	Activated/memory B-cells
CD44	Mouse	IM7	Brilliant Ultraviolet 737	T-cell subsets
CD278	Mouse	7E.17G9	Brilliant Violet 421	T Follicular helper/Treg
PD-1	Mouse	J43	Brilliant Violet 480	T Follicular helper cells
FVS575V	All Species		FVS575V	Viable cells
CD80	Mouse	16-10A1	Brilliant Violet 650	Activated B-cells
IgM	Mouse	R6-60.2	Brilliant Violet 711	B-cell/ASC subsets
CD4	Mouse	RM4-5	Brilliant Violet 786	CD4+ T-cells
CD19	Mouse	1D3	Brilliant Violet 786	B-cell subsets
CD138	Mouse	281-2	Brilliant Blue 515	Plasmablasts/Plasma cells
TCR beta	Mouse	H57-597	Brilliant Blue 700	Pan T-cells
FoxP3	Mouse	FJK-16s	PE	Regulatory T-cells
CD45R	Mouse	RA3-6B2	PE-CF594	B-cell subsets
CD25	Mouse	PC61	PE-Cy5	Activated T-cells
CD185	Mouse	2G8	PE-Cy7	T Follicular helper cells
MHC Class II (I-A/I-E)	Mouse	M5/114.15.2 (also known as M5/114)	Alexa Fluor 647	B-cell subsets
CD62L	Mouse	Mel-14	APC-R700	T-cell subsets
CD45	Mouse	30-F11	APC-Cy7	Pan leukocyte

OMIP-075

A 22-color panel for the measurement of antigen-specific T-cell responses in human and nonhuman primates

Cytometry PART A, Volume 99, Issue 9, 884-887 (2021)

Takuto Nogimori, Eiko Moriishi, Mami Ikeda, Shokichi Takahama, Takuya Yamamoto

Description: We aimed to design a multicolor panel to detect antigen-specific CD4+ T cells in non-human 4 primate models (NHPs), for vaccine-related and infectious disease-related studies...

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Live/Dead Fix Blue	All Species		Live/Dead Fix Blue	Viability
CD3	Human	SP34-2	Brilliant Ultraviolet 615	Surface Staining
CD4	Human	S3.5	PE-Cy5.5	Surface Staining
CD8	Human	RPA-T8	Brilliant Ultraviolet 563	Surface Staining
CD45RO	Human	UCHL1	Brilliant Ultraviolet 805	Surface Staining
CD27	Human	1A4CD27	PE-Cy5	Surface Staining
CD28	Human	CD28.2	Brilliant Ultraviolet 805	Surface Staining
CD95	Human	DX2	PE-Cy5	Surface Staining
PD-1	Human	EH12.2H7	Brilliant Violet 750	Surface Staining
CD278	Human	C398.4A	Brilliant Ultraviolet 395	Surface Staining
CD183	Human	G025H7	PE	Surface 37˚C Staining
CD185	Human	MU5UBEE	PE-eFluor 610	Intracellular Staining
CD194	Human	L291H4	Brilliant Violet 510	Surface 37˚C Staining
CCR6	Human	G034E3	APC-Fire 750	Surface 37˚C Staining
CD40L	Human	TRAP1	FITC	Intracellular Staining
IL-2	Human	MQ1-17H12	Brilliant Ultraviolet 737	Intracellular Staining
IL-4	Human	8D4-8	PE-Cy7	Intracellular Staining
IL-13	Human	JES10-5A2	Brilliant Violet 421	Intracellular Staining
IL-17A	Human	BL168	Brilliant Violet 605	Intracellular Staining
IL-21	Human	3A3-N2.1 (also known as 3A3-N2)	Alexa Fluor 647	Intracellular Staining
MIP-1 beta	Human	D21-1351	Alexa Fluor 700	Intracellular Staining
IFN-gamma	Human	4S.B3	Brilliant Violet 786	Intracellular Staining
TNF alpha	Human	MAb11	Brilliant Violet 650	Intracellular Staining
CD107a	Human	H4A3	Brilliant Violet 711	Co-culture

OMIP-074

Phenotypic analysis of IgG and IgA subclasses on human B cells

Cytometry PART A, Volume 99, Issue 9, 880-883 (2021)

Leonard Nettey, Reid Ballard, Thomas Liechti, Rosemarie D. Mason

Description: This panel was designed and optimized to characterize the phenotypic diversity of circulating human memory B cells with an emphasis on discriminating IgA and IgG subclasses.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD19	Human	HIB19	PE-Dazzle 594	Lineage
CD20	Human	2H7	Alexa Fluor 700	B-Cell Subset
CD27	Human	O323	Brilliant Violet 650
CD21	Human	B-ly4	Brilliant Violet 605
IgA	Human		DyLight 405	Immunoglobulin Isotype
IgD	Human		PE-Cy5.5
IgE	Human	MHE-18	APC-Fire 750
IgG	Human	G18-145	PE-Cy5
IgM	Human	MHM-88	Brilliant Violet 570
IgA1	Human	B3506B4	PerCP-Cy5.5	Immunoglobulin subclass
IgA2	Human	IS11-21E11	PE-Vio770
IgG1	Human	HP6001	PE
Biotin	Human		Brilliant Violet 750
IgG4	Human		APC
Ig Kappa Light Chain	Human	G20-193	Brilliant Violet 711	Immunoglobulin light chain
Ig Lambda Light Chain	Human	JDC-12	Brilliant Violet 786
Live/Dead Fix Aqua	Human		Live/Dead Fix Aqua	Viability
IgG2	Human	HP6002	Biotin
IgG3	Human	HP6050	Alexa Fluor 488

OMIP-073

Analysis of human thymocyte development with a 14-color flow cytometry panel

Cytometry PART A, Volume 99, Issue 9, 875-879 (2021)

Sarah-Jolan Bremer, Laura Glau, Christina Gehbauer, Annika Boxnick, Daniel Biermann, Jörg Siegmar Sachweh, Eva Tolosa, Anna Gieras

Description: This panel was designed for the identification and detailed characterization of the different developmental steps of human thymocytes. We optimized the panel for fresh tissue in order to provide an unbiased analysis of T cell development. Accurate selection of antibodies and precise gating allow us to phenotype 14 major stages of human thymocyte development and illustrate the trajectories of T cell development from early thymic progenitors (ETP) to mature T cells that are ready to populate the periphery. The panel identifies ETPs, T-lineage-committed cells (TC), CD34-positive immature single-positive CD4 cells (ISP4 CD34+), CD34-negative immature single-positive CD4 cells (ISP4 CD34-), CD45-low early double-positive cells (EDP CD45low), CD45-high early double-positive cells (EDP CD45high), late double-positive cells (LDP), single-positive CD4 cells (SP4), single-positive CD8 cells (SP8), ready-to-egress single-positive CD4 cells (rSP4), ready-to-egress single-positive CD8 cells (rSP8), T γδ cells (Tγδ), T regulatory cells (Treg), and ready-to-egress T regulatory cells (rTreg). To highlight important checkpoints during T cell development, we added antibodies relevant for specific developmental steps to the panel. These include CD1a to define TCs, CD28 as a marker for ß-selection and CD69 in combination with CD45RA to determine the maturation stage of thymocytes shortly before they become ready to egress the thymus and colonize the periphery. Moreover, Annexin V, as a marker for apoptosis, provides valuable extra information concerning the apoptotic death of thymocytes. Currently, we use this panel to identify aberrations in T cell development in health and disease.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead Cell	All Species		Alexa Fluor 750	viability
CD25	Human	BC96	Brilliant Violet 421	T regulatory cells
CD45	Human	HI30	Brilliant Violet 510	Leucocytes
CD3	Human	OKT3	Brilliant Violet 650	T cells
CD45RA	Human	HI100	Brilliant Violet 711	Lineage marker during early and late developmental stages
CD8a	Human	RPA-T8	Brilliant Violet 785	CD8 T cells and double-positive cells
CD1a	Human	HI149	FITC	T cell lineage commitment
CD69	Human	FN50	PerCP-Cy5.5	Tissue retention marker
CD34	Human	563	PE	Hematopoietic stem and progenitor cells
CD4	Human	RPA-T4	PE-Dazzle 594	CD4 T cells, ISP4 and double-positive cells
CD28	Human	CD28.2	PE-Cy7	ß-selection
Annexin V	All Species		Alexa Fluor 647	Apoptosis
CD7	Human	M-T701	Alexa Fluor 700	T cell lineage
TCR gamma/delta	Human	11F2	Brilliant Violet 605	T γδ cells

OMIP-072

A 15-color panel for immunophenotypic identification, quantification, and characterization of leukemic stem cells in children with acute myeloid leukemia

Cytometry PART A, Volume 99, Issue 4, 382-387 (2021)

Marianne A. Petersen, Marie Bill, Carina A. Rosenberg

Description: This panel was designed to identify, quantify and phenotypically characterize putative leukemic stem cells (LSCs) in bone marrow (BM) samples from individual pediatric patients diagnosed with acute myeloid leukemia (AML). Based on an aberrant expression on immunophenotypically defined hematopoietic stem cells (HSCs), several antigens have been proposed as LSC markers in AML research, using healthy adult BM samples as reference material. Generally, these antigens have been evaluated individually in smaller panels (e.g. 8-color panels). This necessitates several tubes to characterize the LSC phenotype and compromises the ability to evaluate LSC heterogeneity. The present 15-color OMIP incorporates nine suggested LSC markers to comprehensively capture LSC immunophenotypes and to explore heterogenic marker-patterns within LSC populations in a single tube. Importantly, this single tube approach requires less input material, which is essential when sampling BM aspirates from pediatric patients where sample volumes often are sparse. As knowledge on normal expression levels of the included LSC markers in HSCs from hematologically healthy children are a prerequisite for labelling a phenotype as abnormal, we have evaluated the applicability of the panel on cryopreserved mononuclear cells (MNCs) isolated from BM samples from pediatric patients without hematological disorders as well as pediatric AML patients. The panel is optimized for cryopreserved BM MNCs, but could in principle, be utilized for LSC detection in any biological material containing human hematopoietic cells.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CLEC12A	Human	HB3	PE
IL-1 RAcP	Human	REA558	VioBright FITC
Bcl-2	Human	bcl-2/100	V450
CD45	Human	HI30	Brilliant Violet 510
CD117	Human	104D2	Brilliant Violet 650
TIM-3	Human	7D3	Brilliant Violet 711
CD38	Human	HIT2	Brilliant Violet 786
CD34	Human	581	PerCP-Cy5.5
CD45RA	Human	HI100	PE-CF594
CD3	Human	SK7	PE-Cy5.5
Dead Cell	All Species		Zombie Red	viability
CD123	Human	7G3	PE-Cy7
CD93	Human	VIMD2	APC
CD25	Human	2A3	APC-R700
CD99	Human	3B2/TA8	APC-Vio770

OMIP-071

A 31-Parameter Flow Cytometry Panel for In-Depth Immunophenotyping of Human T-Cell Subsets Using Surface Markers

Cytometry PART A, Volume 99, Issue 3, 273-277 (2021)

Song-Rong Wang, Na Zhong, Xin-Mei Zhang, Zhi-Bin Zhao, Robert Balderas, Liang Li, Zhe-Xiong Lian

Description: Dissecting the functional diversity of T cells is critical in elucidating mechanisms and in developing therapies for various diseases. Here, we designed a 31-parameter (29-color) panel to enable the characterization of T-cell subsets and immunophenotyping of the human peripheral blood and lymph nodes using cell surface staining. In addition to adaptive T-cell markers, TCR Vα24-Jα18, TCR γδ, TCR Vɑ7.2, and CD161 were included to identify iNKT, γδ T, and MAIT cells, respectively, which are innate-like T cells. C-X-C chemokine receptors (CXCR3, CXCR4, CXCR5, CXCR6) and C-C motif chemokine receptors (CCR4, CCR6, CCR7) were included to enable the identification of Th cell subsets (Th1, Th2, Th17), Tfh cell subsets (Tfh1, Tfh2, Tfh17), and Th cells with specific homing capacities. Furthermore, in this panel, we also used markers for assessing cell differentiation (CD45RO, CD7), activation (CD57, CD95, HLA-DR) and the expression of some cosignaling molecules (PD-1, NKG2D, CD28). Particularly, CD69 and CD103 were included for the further analysis of tissue resident memory T (Trm) cells. This panel would enable the in-depth immunophenotyping of human T-cell subsets, and may be applied in the monitoring, prognosis, and mechanistic studies of various immune-related diseases.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD185	Human	RF8B2	Brilliant Ultraviolet 615
TCR V alpha 7.2	Human	OF-5A12	Brilliant Violet 570
CD57	Human	NK-1	Brilliant Violet 480
CD103	Human	2E7	Brilliant Blue 630-P2
CD314	Human	1D11	Brilliant Blue 660-P2
CD194	Human	[1G1]	Brilliant Ultraviolet 395
CD95	Human	DX2	Brilliant Ultraviolet 395
CCR7	Human	2-L1-A	APC-Cy7
CD69	Human	FN50	Brilliant Blue 790-P
CD127	Human	HIL-7R-M21	PE-Cy5
Dead Cell	All Species		Zombie UV	viability
CD3	Human	UCHT-1	Brilliant Ultraviolet 805
CD4	Human	SK3	Brilliant Violet 750
CD7	Human	M-T701	PE-Cy7
CD8	Human	RPA-T8	Brilliant Violet 786
CD25	Human	2A3	Brilliant Violet 421
CD28	Human	CD28.2	PE
CD45	Human	HI30	APC-R700
CD45RO	Human	UCHL1	PE-CF594
CD56	Human	NCAM16.2	Brilliant Violet 650
CD161	Human	DX12	Brilliant Ultraviolet 737
PD-1	Human	EH12.1	Brilliant Blue 515
HLA-DR	Human	G46-6	Brilliant Violet 605
TCR delta/gamma	Human		APC
CD183	Human	G025H7	Brilliant Violet 510
CXCR4	Human	12G5	Brilliant Ultraviolet 563
CD186	Human	13B 1E5	Brilliant Blue 700
CCR6	Human	11A9	Brilliant Ultraviolet 496
TCR V alpha 24 J alpha 18	Human	6B11	Brilliant Violet 711

OMIP-070

NKp46‐Based 27‐Color Phenotyping to Define Natural Killer Cells Isolated From Human Tumor Tissues

Cytometry PART A, Volume 97, Issue 10, 1052-1056 (2020)

Marie Frutoso, Florian Mair, Martin Prlic

Description: This 27‐color panel has been validated and optimized to comprehensively profile natural killer (NK) cells isolated from human tumors using a collagenase Type II‐based digestion protocol. We confirmed that detection of protein expression by antibodies used in our final panel was not affected during tissue digestion. During this evaluation process, we found that detection of CD56, a biomarker typically used to identify NK cells, was affected substantially by collagenase‐based digestion. Thus, our panel is centered around expression of NKp46, which is sufficient to identify NK cells and not affected by the tissue collagenase digestion step. Our panel further includes biomarkers used to extrapolate NK‐cell maturation, differentiation, migration, homing potential, and functional state. Our panel is intended to provide in‐depth characterization of human NK cells isolated from tissues, which we specifically tested using oral squamous cell carcinomas tissues, but it is compatible with other tissues that can be dissociated with a collagenase Type II‐based protocol.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Blue	viability
Streptavidin	Human		Brilliant Blue 630-P2	secondary reagent to NKp46
CD27	Human	M-T271	Brilliant Blue 660-P2	maturation
CD38	Human	HIT2	Brilliant Blue 790-P	activation
CD45	Human	HI30	Brilliant Ultraviolet 805	lymphocyte
CD14	Human	M5E2	Brilliant Violet 570	monocyte
CD19	Human		Brilliant Ultraviolet 395	B
CD3	Human	UCHT1	Brilliant Ultraviolet 661	T
CD127	Human	hIL-7R-M21	Brilliant Violet 786	ILC
HLA-DR	Human		APC-H7	exclusion/activtion
CD16	Human	3G8	Brilliant Ultraviolet 496	NK and ADCC
NKp46	Human	900	Biotin	NK and activation
CD314	Human	1D11	PE-Cy7	activation
CD244	Human	25235	Brilliant Violet 421	activation
CD2	Human	RPA-2.10	Brilliant Violet 605	co-stimulatory
CD159a (NKG2a)	Human	REA110	PE	inhibitory
CD161	Human	DX12	Brilliant Blue 700	inhibitory
TIGIT	Human	741182	Brilliant Violet 711	co-inhibitory
CD69	Human	FN50	Brilliant Violet 650	residency and activation
CD39	Human	TU66	PE-CF594	residency and activation
CD57	Human	NK-1	FITC	maturation and differentiation
CD11b	Human	ICRF44	PE-Cy5	maturation and adhesion
CX3CR1	Human	2A9-1	Brilliant Ultraviolet 737	migration
CD103	Human	Ber-ACT8	Brilliant Violet 750	residency
Ki-67	Human	SolA15	eFluor 660	proliferation
Granzyme B	Human		Alexa Fluor 700	cytolytic ability
CD25	Human	2A3	Brilliant Ultraviolet 563	activation
CD56	Human	CMSSB	PE-Cy5.5	activation, NK for PB

OMIP-069

Forty‐Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood

Cytometry PART A, Volume 97, Issue 10, 1044-1051 (2020)

Lily M. Park, Joanne Lannigan, Maria C. Jaimes

Description: This 40‐color flow cytometry‐based panel was developed for in‐depth immunophenotyping of the major cell subsets present in human peripheral blood. Sample availability can often be limited, especially in cases of clinical trial material, when multiple types of testing are required from a single sample or timepoint. Maximizing the amount of information that can be obtained from a single sample not only provides more in‐depth characterization of the immune system but also serves to address the issue of limited sample availability. The panel presented here identifies CD4 T cells, CD8 T cells, regulatory T cells, γδ T cells, NKT‐like cells, B cells, NK cells, monocytes and dendritic cells. For each specific cell type, the panel includes markers for further characterization by including a selection of activation and differentiation markers, as well as chemokine receptors. Moreover, the combination of multiple markers in one tube might lead to the discovery of new immune phenotypes and their relevance in certain diseases. Of note, this panel was designed to include only surface markers to avoid the need for fixation and permeabilization steps. The panel can be used for studies aimed at characterizing the immune response in the context of infectious or autoimmune diseases, monitoring cancer patients on immuno‐ or chemotherapy, and discovery of unique and targetable biomarkers. Different from all previously published OMIPs, this panel was developed using a full spectrum flow cytometer, a technology that has allowed the effective use of 40 fluorescent markers in a single panel. The panel was developed using cryopreserved human peripheral blood mononuclear cells (PBMC) from healthy adults (Table 1). Although we have not tested the panel on fresh PBMCs or whole blood, it is anticipated that the panel could be used in those sample preparations without further optimization.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: Cytek Aurora 5 Laser

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Blue	Viability
CD4	Human	SK3	cFluor YG584	CD4 T and NKT cells
CD45	Human	2D1	PerCP	Leukocyte
CD3	Human	SK7	Brilliant Violet 510	Pan T cell, NKT cells
CD8	Human	SK1	Brilliant Ultraviolet 805	CD8 T, NK, and NKT cells
CD25	Human	CD25-3G10	PE-Alexa 700	Regulatory T cells
TCR gamma/delta	Human	B1.1	PerCP-eFluor 710	Pan ?d T cell
CD14	Human	63D3	Spark Blue 550	Monocyte differentiation
CD16	Human	3G8	Brilliant Ultraviolet 496	Monocyte, NK cell, and Dendritic cell differentiation
CD11c	Human	3.9	eFluor 450	Dendritic Cell differentiation
CD19	Human	HIB19	Spark NIR 685	B cells
CD20	Human	HI47	Pacific Orange	B cells
CD24	Human	SN3	PE-Alexa 610	B cell differentiation
CD39	Human	TU66	Brilliant Ultraviolet 661	B cell, T reg, and monocyte differentiation
IgD	Human	IA6-2	Brilliant Violet 480	B cell differentiation
IgG	Human	G18-145	Brilliant Violet 605	B cell differentiation
IgM	Human	MHM-88	Brilliant Violet 570	B cell differentiation
CD141	Human	1A4	Brilliant Blue 515	Dendritic cell differentiation
CD1c	Human	L161	Alexa Fluor 647	Dendritic cells, NKT cells
CD123	Human	6H6	SuperBright 436	Plasmacytoid dendritic cells
CD2	Human	TS1/8	PerCP-Cy5.5	NK cell differentiation
CD56	Human	NCAM16.2	Brilliant Ultraviolet 737	Pan NK cell, ?d T cell activation
ccr7	Human	G043H7	Brilliant Violet 421	T cell differentiation
CD27	Human	M-T271	APC-H7	T and B cell differentiation
CD28	Human	CD28.2	Brilliant Violet 650	T cell and NK cell differentiation
CD45RA	Human	5H9	Brilliant Ultraviolet 395	T cell and dendritic cell differentiation
CD95	Human	Dx2	PE-Cy5	T cell and B cell differentiation
CD127	Human	hIL-7R-M21	APC-R700	Cytokine receptor; T cell differentiation
CD337	Human	p30-15	PE-Dazzle 594	NK cell differentiation
CCR6	Human	G034E3	Brilliant Violet 711	Chemokine receptor; T cell and B cell differentiation
CCR5	Human	2D7/CCR5	Brilliant Ultraviolet 563	Chemokine receptor; Monocyte, dendritic cell, T cell, and B cell differentiation
CD185	Human	RF8B2	Brilliant Violet 750	Chemokine receptor; T cell differentiation
CD183	Human	G025H7	PE-Cy7	Chemokine receptor; Dendritic cell, T cell, and B cell differentiation
CD38	Human	HIT2	APC-Fire 810	Monocyte, dendritic cell, T cell, and B cell activation/differentiation
CD57	Human	HNK-1	FITC	NK and CD8+ T cell immune senescence
PD-1	Human	EH12.2H7	Brilliant Violet 785	T cell inhibitory receptor
CD159a (NKG2a)	Human	REA110	APC	NK, NKT, and ?d T cell activation/differentiation
CD159c	Human	REA205	PE	NK cell differentiation
CD314	Human	1D11	Brilliant Ultraviolet 615	NK cell differentiation

OMIP-068

High‐Dimensional Characterization of Global and Antigen‐Specific B Cells in Chronic Infection

Cytometry PART A, Volume 97, Issue 10, 1037-1043 (2020)

Katherine Cascino, Mario Roederer, Thomas Liechti

Description: This 24‐color flow cytometry panel focuses on characterizing antigen‐specific B cells and precise delineation of B‐cell subsets in chronic infections and is applicable to other chronic diseases such as autoimmunity. The panel was optimized for human cryopreserved peripheral blood mononuclear cells (PBMCs). Markers were chosen to extensively distinguish B‐cell lineages (CD19, CD20, CD10, CD38, CD24, IgM, IgD, CD27, CD21, CD43, CD5). Inclusion of antigen‐specific probes was of high priority in order to assess hepatitis B virus (HBV) antigen‐specific B cells for our purposes. These probes can be readily exchanged for other pathogen‐specific probes or additional markers for the panel to be tailored to desired research questions beyond HBV. In addition, we included a comprehensive and unique set of functional markers such as chemokine receptors (CXCR3, CXCR5), co‐stimulatory molecule (CD86), Fc receptor (CD32), regulatory molecules (BTLA, CD39), and inhibitory markers associated with chronic infections (PD‐1, FcRL5, CD11c, CD22) to enable in‐depth analysis of global and antigen‐specific B cells during chronic infection.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Aqua	n/a
CD11c	Human	Bu15	Brilliant Blue 630-P2	n/a
FCRL5	Human	509f6	Brilliant Blue 660-P2	n/a
IgD	Human	IA6-2	Brilliant Blue 790-P	n/a
CD21	Human	B-ly4	Brilliant Ultraviolet 496	n/a
CD86	Human		Brilliant Ultraviolet 737	n/a
PD-1	Human	EH12.2H7	FITC	n/a
CD272	Human	J168-540	Brilliant Blue 700	n/a
CD27	Human	M-T271	PE-CF594	n/a
CD10	Human	HI10a	PE-Cy5	n/a
CD5	Human	CD5-5D7	PE-Cy5.5	n/a
CD185	Human	J252D4	PE-Cy7	n/a
IgM	Human		Alexa Fluor 700	n/a
CD19	Human		APC-H7	n/a
CD24	Human	ML5	Brilliant Ultraviolet 395	n/a
CD38	Human	HIT2	Brilliant Ultraviolet 661	n/a
CD22	Human	HIB22	Brilliant Ultraviolet 805	n/a
CD183	Human	G025H7	Brilliant Violet 421	n/a
CD3	Human	UCHT1	Brilliant Violet 510	n/a
CD14	Human	MfP9	Brilliant Violet 510	n/a
CD43	Human	1G10	Brilliant Violet 605	n/a
CD39	Human	TU66	Brilliant Violet 711	n/a
CD32	Human	3D3	Brilliant Violet 750	n/a
CD20	Human		Brilliant Violet 786	n/a

OMIP-067

28‐Color Flow Cytometry Panel to Evaluate Human T‐Cell Phenotype and Function

Cytometry PART A, Volume 97, Issue 10, 1032-1036 (2020)

Phillip A. Swanson II, Robert A. Seder

Description: This 28-color flow cytometry panel was developed to detect frequencies and function of a broad spectrum of T-cell subsets from the blood of infants and adults following antigen-specific stimulation. This panel includes markers to characterize αβ and γδ T-cell subsets, T regulatory cells (Tregs), T follicular helper cells (TfH), and recent thymic emigrants (RTEs). Additionally, antibodies that measure cell surface activation molecules (CD154, CD69, HLA-DR, and CD38) and function through detection of cytokines (IL-8, IL-2, IL-4, IL-5, IL-13, IL-21, IFNγ, and TNF) are also included. This panel was optimized using PMA-stimulated cryopreserved healthy adult and infant PBMCs as well as stimulation with CMV-specific peptides. Currently, this panel is being used to evaluate antigen-specific T-cell responses in adults and infants following immunization with an attenuated malaria vaccine (Table 1).

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Blue	Viability
CD69	Human	FN50	Brilliant Blue 790-P	Activation
IL-8	Human	G265-8	Brilliant Blue 630-P2	Function
PD-1	Human	EH12.2H7	Brilliant Blue 660-P2	CO-inhibitory
ccr7	Human	G043H7	Alexa Fluor 700	Differentiation
CD3	Human	UCHT1	Brilliant Ultraviolet 496	Lineage
CD4	Human	SK3	Brilliant Ultraviolet 805	Lineage
CD8	Human	RPA-T8	PE	Lineage
CD25	Human	BC96	Brilliant Blue 700	Differentiation
CD27	Human	O323	Brilliant Violet 785	Differentiation
CD31	Human	WM59	Brilliant Violet 711	Differentiation
CD38	Human	HIT2	Brilliant Ultraviolet 661	Activation
CD45RA	Human		Brilliant Violet 570	Differentiation
CD127	Human	A019D5	PE-Cy7	Differentiation
CD40L	Human	24-31	Brilliant Violet 605	Co-stimulatory
CX3CR1	Human	2A9-1	Brilliant Violet 650	Differentiation
CD183	Human	G025H7	PE-Dazzle 594	Trafficking
CD185	Human	RF8B2	Brilliant Ultraviolet 563	Trafficking
HLA-DR	Human	Tu36	PE-Cy5.5	Activation
IFN-gamma	Human	B27	Brilliant Ultraviolet 395	Function
IL-2	Human	MQ1-17H12	Brilliant Ultraviolet 737	Function
IL-4	Human	MP4-25D2	Brilliant Violet 421	Function
IL-5	Human	TRFK5	Brilliant Violet 421	Function
IL-13	Human	JES10-5A2	Brilliant Violet 421	Function
IL-21	Human	3A3-N2.1	Alexa Fluor 647	Function
TCR gamma/delta	Human	REA591	APC-Vio770	Lineage
TCR V gamma 9	Human		PE-Cy5	Lineage
TCR V delta 1	Human	TS8.2	FITC	Lineage
TCR V delta 2	Human	123R3	VioGreen	Lineage
TNF alpha	Human	Mab11	Brilliant Violet 750	Function

OMIP-066

Identification of Novel Subpopulations of Human Group 2 Innate Lymphoid Cells in Peripheral Blood

Cytometry PART A, Volume 97, Issue 10, 1028-1031 (2020)

Yoichiro Ohne

Description: This 14‐color flow cytometry panel was designed to identify newly described subpopulations within human group 2 innate lymphoid cells (ILC2s) and other ILC subsets. This panel also allowed to identify recently reported subpopulations of peripheral blood CRTH2− c‐Kit+ ILCs. We validated this panel mostly in human peripheral blood but also confirmed that the same panel and gating strategy works well in human tonsillar cells. The panel contains a few markers indicating the activation status of ILCs. In addition, phycoerythrin (PE) channel is available for the markers of interest in each study. In the validation studies described here, PE channel was used to test the expression of some markers. These features make this panel applicable for immunophenotyping of ILCs in various disease states. © 2020 International Society for Advancement of Cytometry

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Blue	Viability
KLRG1	Human	13F12F2	SuperBright 702	Subpopulation
CD56	Human	NCAM16.2	APC-R700	Subpopulation
CCR10	Human	1B5	Brilliant Ultraviolet 395	Subpopulation
CD45	Human	HI30	Brilliant Ultraviolet 805	Lymphocyte
CD3	Human	SK7	Brilliant Violet 510	Lineage/Dump
CD4	Human	RPA-T4	Brilliant Violet 510	Lineage/Dump
CD14	Human	M5E2	Brilliant Violet 510	Lineage/Dump
CD19	Human		Brilliant Violet 510	Lineage/Dump
CD34	Human	581	Brilliant Violet 510	Lineage/Dump
CD123	Human	6H6	Brilliant Violet 510	Lineage/Dump
CCR6	Human	G034E3	Brilliant Violet 421	Lineage/Dump
CD336	Human	p44-8	Brilliant Violet 786	ILC3 in tonsil
CD127	Human	REA614	VioBright 515	Total ILC
TSLP Receptor	Human	1F11	Brilliant Blue 700	ILC2 activation
NKp46	Human	DX22	PE-Dazzle 594	Subpopulation
CD117	Human	104D2	PE-Cy7	Subpopulation
CD294	Human	BM16	Alexa Fluor 647	ILC2
CD94	Human		APC-Fire 750	NK

OMIP-065

Dog Immunophenotyping and T‐Cell Activity Evaluation with a 14‐Color Panel

Cytometry PART A, Volume 97, Issue 10, 1024-1027 (2020)

Stanislav Pantelyushin, Elisabeth Ranninger, Regula Bettschart‐Wolfensberger, Johannes vom Berg

Description: The purpose of the panel described here is to assess the immune cell composition and their functionality in the peripheral blood mononuclear cells (PBMCs) of dogs. Moreover, its “plug and play” composition allows for an in‐depth analysis of T‐cell responses in ex vivo assays (Table 1). Initially, this panel has been designed for the analysis of cryopreserved PBMCs to allow batched analysis and to reduce interexperimental variation. Withers and colleagues published a comparable and—to our knowledge—currently the most extensive canine panel to date (1). While their study focused on the aging and activation status of T cells in dogs, our panel is designed to look at a broader range of cells with a higher number of markers. This allows a more in‐depth analysis of functional extracellular and intracellular markers. In addition, all antibodies in our proposed panel are directly labeled. In combination with suitable lymphocyte isolation protocols, this panel could potentially also be adapted to analyze tissue biopsies from various different organs.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD45	Dog	YKIX716.13	eFluor 450	Leukocyte
CD25	Dog	P4A10	SuperBright 600	Treg, activation
Dead cells	Dog		Zombie Aqua	viability
CD4	Dog	YKIX302.9	SuperBright 645	CD4+ T
CD8a	Dog	YCATE55.9	SuperBright 702	CD8+ T
CD14	Dog	M5E2	Brilliant Violet 785	Monocyte
CD3	Dog	CA17.2A12	FITC	T
EOMES	Dog	WD1928	PerCP-eFluor 710	CD8+ T transcription factor
CD22	Dog	RFB-4	PE	B
Granzyme B	Dog	GB11	PE-CF594	cytotoxic activation, NK
FoxP3	Dog	FJK-16s	PE-Cy7	Treg
MHC II	Dog	YKIX334.2	APC	APC
Ki-67	Dog	SolA15	Alexa Fluor 700	proliferation
CD5	Dog	YKIX322.3	APC-eFluor 780	T, NK

OMIP-064

A 27‐Color Flow Cytometry Panel to Detect and Characterize Human NK Cells and Other Innate Lymphoid Cell Subsets, MAIT Cells, and γδ T Cells

Cytometry PART A, Volume 97, Issue 10, 1019-1023 (2020)

Nina Hertoghs, Katharine V. Schwedhelm, Kenneth D. Stuart, Margaret Juliana McElrath, Stephen C. De Rosa

Description: This 27‐color flow cytometry panel was developed in order to assess immunological changes over the course of an immunization and challenge regimen in two experimental malaria vaccine trials. The aim of the study was to find correlates of vaccine‐induced protection. Several studies have indicated that protection against malaria appears to involve immune responses at various immunological sites, with liver‐resident responses playing an essential role. As it is not feasible to monitor the immune responses within the liver in humans, this panel is developed with the aim to thoroughly characterize the immune responses over time in blood in addition to detecting changes that might reflect what happens in other immunological sites like the liver. The focus of this panel is to detect several innate lymphoid cell populations, including NK cells and their activation status. Moreover, unconventional T cells like mucosal associated invariant T cells and γδ T cells are assessed in the panel.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD159c	Human	134591	Alexa Fluor 700	NK phenotyping
Ki-67	Human	B56	Brilliant Blue 660-P2	Proliferation
Fc epsilon R1 alpha	Human	polyclonal	FITC	NK phenotyping
CD57	Human	HNK-1	Brilliant Violet 711	NK phenotyping
CD127	Human	hIL-7R-M21	Brilliant Blue 790-P	ILC phenotyping
CD16	Human	3G8	Brilliant Ultraviolet 395	NK, monocyte, ILC
Dead cells	Human		Live/Dead Fix Blue	Viability
CD3	Human	UCHT1	Brilliant Ultraviolet 496	T cell
CD19	Human	SJ25C1	Brilliant Ultraviolet 563	B cell
HLA-DR	Human	G46-6	Brilliant Ultraviolet 661	Activation
CD27	Human	L128	Brilliant Ultraviolet 737	NK phenotyping
CD8	Human	SK1	Brilliant Ultraviolet 805	CD8 T CELL
TCR gamma/delta	Human	11F2	Brilliant Violet 421	Pan-?d T
CD314	Human	1D11	Brilliant Violet 480	NK phenotyping
CD56	Human	HCD56	Brilliant Violet 570	NK phenotyping
TCR V delta 2	Human	B6	Brilliant Violet 605	?d T cell subset vd2
CD38	Human	HB-7	Brilliant Violet 650	Activation
CD4	Human	SK3	Brilliant Violet 750	CD4 T
TCR V alpha 7.2	Human	3C10	Brilliant Violet 786	MAIT
CD14	Human	MfP9	PerCP-Cy5.5	Monocyte
NKp46	Human	9E2/NKp46	PE	NK phenotyping
CD294	Human	BM16	PE-CF594	ILC phenotyping
CD33	Human	WM53	PE-Cy5	Granulocyte/monocyte exclusion
CD117	Human	104D2	PE-Cy5.5	ILC phenotyping
CD159a (NKG2a)	Human	Z199	PE-Cy7	NK phenotyping
CD337	Human	p30-15	Alexa Fluor 647	NK phenotyping
CD161	Human	HP-3G10	APC-Fire 750	MAIT/ILC phenotyping

OMIP-063

28‐Color Flow Cytometry Panel for Broad Human Immunophenotyping

Cytometry PART A, Volume 97, Issue 8, 777-781 (2020)

Kathryn Payne, Wenyan Li, Robert Salomon, Cindy S. Ma

Description: A 28-color panel was developed to screen for a range of lymphocyte subsets in human peripheral blood mononuclear cells (PBMCs), particularly in patients with primary immunodeficiency (PID). Using the panel, we are able to avoid running the sample over multiple screening panels while still deeply phenotyping a diverse range of lymphocyte subsets including innate like lymphocytes (γδ, mucosal-associated invariant T [MAIT], natural killer [NK], and NKT cells), as well as multiple subsets of naïve and memory CD4+ and CD8+ T cells, and B cells. Specifically, naïve, central memory (cmem) and effector memory (emem) CD4+ T cells, naïve cmem, emem, and CD45RA+ revertant memory (TEMRA) CD8+ T cells, regulatory (Tregs), T follicular helper (Tfh) and T helper (Th) 1, and Th17 CD4+ T cells, and transitional, naïve, memory, and CD21 B cells.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD185	Human	RF8B2	Brilliant Ultraviolet 615	Tfh
CD19	Human	SJ25C1	Brilliant Violet 750	B Cell
CD21	Human	B-ly4	Brilliant Ultraviolet 563	B Cell (Down-regulated on atypical/aged memory B cells)
CD56	Human	NCAM16.2	Brilliant Blue 790-P	NK cells and some memory CD8+ T cells
IgG	Human	G18-145	Brilliant Blue 660-P2	IgG+ Isotype switched memory B cells
CCR6	Human	11A9	Brilliant Blue 630-P2	Th17
IgA1/IgA2	Human	G20-359	PE-Cy5	IgA+ Isotype switched memory B cells
CD25	Human	2A3	BYG584-P	Treg
IgM	Human	G20-127	APC-R700	Naïve B cells and IgM+ memory B cells
Dead cells	Human		Zombie UV	Viability
CD20	Human	2H7	Brilliant Ultraviolet 805	B Cell
TCR alpha/beta	Human	T10B9.1A-31	Brilliant Ultraviolet 737	T cell (conventional aß T)
CD4	Human	SK3	Brilliant Ultraviolet 661	T helper
CD8	Human	RPA-T8	Brilliant Ultraviolet 496	Cytotoxic T cell
CD45RA	Human	HI100	Brilliant Ultraviolet 395	naïve and revertant effector (TEMRA) T cells
CD161	Human	DX12	Brilliant Violet 786	MAIT, NK, NKT, and a subset of CD8+ T cells
TCR gamma/delta	Human	11F2	Brilliant Violet 711	?d T cells
CD10	Human	HI10a	Brilliant Violet 650	Transitional B cells
CD3	Human	UCHT1	Brilliant Violet 570	T cell
IgD	Human	IA6-2	Brilliant Violet 480	Naive B cells, low levels on IgM memory B cells
CD183	Human	1C6	Brilliant Violet 421	Th1
CD127	Human	hIL-7R-M21	Brilliant Blue 700	Down-regulated on Tregs
CD27	Human	M-T271	Brilliant Blue 515	Memory B cells; naïve and cmem CD4+ and CD8+ T cells
ccr7	Human	G043H7	PE-Cy7	Naïve T and CMEM T cells
PD-1	Human	EH12.1	PE-CF594	Activation
TCR V alpha 7.2	Human	3C10	APC-Cy7	T cell receptor for MAIT cells
KLRG1	Human	2F1/KLRG1	APC	senescence on CD8+ T cells

OMIP-062

A 14‐Color, 16‐Antibody Panel for Immunophenotyping Human Innate Lymphoid, Myeloid and T Cells in Small Volumes of Whole Blood and Pediatric Airway Samples

Cytometry PART A, Volume 95, Issue 12, 1231-1235 (2019)

Dawid Swieboda, Yanping Guo, Sophie Sagawe, Ryan S. Thwaites, Simon Nadel, Peter J.M. Openshaw, Fiona J. Culley

Description: This 14‐color, 16‐antibody OMIP was designed for enumeration of leukocyte responses in pediatric samples, where sample volumes and cell numbers can be very low. Leukocytes identified by this panel include all major members of the innate lymphoid cell (ILC) family (ILC1s, ILC2s, and ILC3s), natural killer cells (NK cells), granulocytes (neutrophils and eosinophils), T‐cells (CD4+ and CD8+), mucosal‐associated invariant T cells (MAIT cells) and NKT‐like cells. The protocol was optimized using small volumes of peripheral blood and validated in airway samples obtained from children (< 2 years of age) admitted to a pediatric intensive care unit (PICU). Given this backdrop, this OMIP is widely applicable to clinical research using low volume or paucicellular samples, such as studies of innate and adaptive immune responses in infants and children, with potential clinical application in diagnostics and monitoring of patients by pediatricians.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD127	Human	A019D5	Brilliant Violet 421	ILCs
CD14	Human	63D3	Brilliant Violet 510	Lineage
CD19	Human	HIB19	Brilliant Violet 510	Lineage
Fc epsilon R1 alpha	Human		Brilliant Violet 510	Lineage
CD123	Human	6H6	Brilliant Violet 510	Lineage
CD4	Human	RPA-T4	Brilliant Violet 605	CD4+ T cells
CD16	Human	3G8	Brilliant Violet 650	NK cells/neutrophils
CD8	Human	SK1	Brilliant Violet 711	CD8+ T cells
TCR V alpha 7.2	Human	3C10	Brilliant Violet 785	MAIT cells
CD45	Human	HI30	FITC	Leukocyte
CD117	Human	A3C6E2	PerCP-Cy5.5	ILC3
CD3	Human	OKT3	PE	T cells
CD161	Human	HP-3G10	PE-Dazzle 594	MAIT cells
CD56	Human	5.1H11	PE-Cy7	NK/NKT-like cells
CD294	Human	BM16	Alexa Fluor 647	ILC2/Th2/Tc2 subsets
CD66b	Human	G10F5	Alexa Fluor 700	Eosinophils
Dead cells	Human		Live/Dead Fix Near IR (775)	Viability

OMIP-061

20‐Color Flow Cytometry Panel for High‐Dimensional Characterization of Murine Antigen‐Presenting Cells

Cytometry PART A, Volume 95, Issue 12, 1226-1230 (2019)

Anthony T. DiPiazza, Juliane P. Hill, Barney S. Graham, Tracy J. Ruckwardt

Description: This 20‐color flow cytometry panel was designed to resolve the cellular heterogeneity of antigen‐presenting cells and was optimized for lymph node tissue. Reagents were carefully selected and optimized for identification of B cells (B220), neutrophils (Ly6G), monocytes and macrophages (Ly6C, CD169, F4/80), and dendritic cells (XCR1, CD172a, CD11c, I‐A/I‐E, CD24, CD64, pDCA‐1, CD103, CD11b). Inclusion of additional functional markers involved in cell migration (CCR7), co‐stimulation (CD80), and adhesion (ICAM‐1) enabled further phenotypic characterization. Finally, this panel has been tested and is compatible with fluorescently labeled antigens such as Alexa Fluor 488 (Ax488) for the study of antigen‐bearing cells in vivo.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Mouse		Live/Dead Fix Blue	Viability
CD45R (B220)	Mouse	RA3-6B2	Brilliant Ultraviolet 496	B cells and pDCs
CD172a	Mouse	P84	PerCP-eFluor 710	cDC2, other myeloid cells
CD11c	Mouse	N418	Brilliant Violet 421	macrophage and DC
CD103	Mouse	M290	Brilliant Violet 510	Lung migratory cDC1
Ly-6C	Mouse	HK1.4	Brilliant Violet 570	Monocytes/macrophages, neutrophils, moDC
CD24	Mouse	M1/69	Brilliant Violet 605	cDC lineage
XCR1	Mouse	ZET	Brilliant Violet 650	pDC (plasmacytoid DC)
MHC Class II (I-A/I-E)	Mouse	M5/114	Brilliant Violet 711	Activation, antigen p
CD3	Mouse	17A2	Brilliant Violet 750	T and NKT cells
CD64	Mouse	X54-5/7.1	Brilliant Violet 786	Monocyte/macrophage, moDC lineage
ccr7	Mouse	4B12	PE	Trafficking
CD80	Mouse	16-10A1	PE-CF594	Co-stimulation
F4/80	Mouse	BM8	PE-Cy5	Macrophage subsets
CD169	Mouse	3D6.112	PE-Cy7	Macrophage subsets
ICAM-1	Mouse		Brilliant Ultraviolet 395	Adhesion
CD45	Mouse	30-F11	Brilliant Ultraviolet 661	Hematopoietic cell lineage
Ly-6G	Mouse	1A8	Brilliant Ultraviolet 737	Neutrophil
CD317	Mouse	927	APC	pDC
CD11b	Mouse	M1/70	APC-R700	Macrophages and dendritic cells

OMIP-060

30‐Parameter Flow Cytometry Panel to Assess T Cell Effector Functions and Regulatory T Cells

Cytometry PART A, Volume 95, Issue 11, 1129-1134 (2019)

Thomas Liechti, Mario Roederer

Description: We developed this comprehensive 28‐color flow cytometry panel with the aim to measure a variety of T cell effector functions in combination with T cell differentiation markers (CCR7, CD27, CD28, CD45RO, CD95) in γδ T cells and CD4+ and CD8+ αβ T cells (Table 1). The effector functions measured in this panel include activation and co‐stimulatory molecules (CD69, CD137, and CD154), cytokines (IL‐2, IL‐13, IL‐17A, IL‐21, IL‐22, TNF, and IFNγ), the chemokine IL‐8, cytotoxic molecules (perforin and granzyme B), and the degranulation marker CD107a. In addition, Ki67 enables the identification and analysis of recently activated T cells. To characterize regulatory T cells (Tregs), we included CD25, CD39, and the canonical Tregs transcription factor FoxP3. We developed and optimized this panel for cryopreserved human peripheral blood mononuclear cells (PBMC) and stimulation with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin. However, we successfully tested other types of stimulation such as staphylococcus enterotoxin B (SEB) or a mix of immunodominant peptides (CEF peptide pool) from cytomegalovirus (CMV), Epstein–Barr virus (EBV) and influenza.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
IL-8	Human	G265-8	Brilliant Blue 630-P2	Effector function
IL-13	Human	JES10-5A2	Brilliant Blue 660-P2	Effector function
CD137	Human	4B4-1	Brilliant Blue 790-P	Co-stimulatory
TCR gamma/delta	Human	B1	PE-Cy5	?dT
ccr7	Human	150503	Brilliant Ultraviolet 395	Differentiation
CD39	Human	TU66	Brilliant Ultraviolet 661	Differentiation
Perforin	Human	B-D48	Alexa Fluor 594	cytotoxic molecule
Dead cells	Human		Live/Dead Fix Blue	viability
Granzyme B	Human	GB11	FITC	Cytotoxic molecule
IFN-gamma	Human	B27	Brilliant Blue 700	Effector function
CD28	Human	CD28.2	PE	Differentiation
FoxP3	Human	PCH101	PE-Cy5.5	Treg
IL-22	Human	22URTI	PE-Cy7	Effector function
IL-21	Human	3A3-N2.1	Alexa Fluor 647	Effector function
CD107a	Human	H4A3	Alexa Fluor 700	Degranulation
CD3	Human	SK7	APC-H7	T cell lineage
CD4	Human	SK3	Brilliant Ultraviolet 496	T cell lineage
CD25	Human	2A3	Brilliant Ultraviolet 563	Treg
CD95	Human	Dx2	Brilliant Ultraviolet 737	Differentiation
CD8	Human	SK1	Brilliant Ultraviolet 805	T cell lineage
IL-2	Human	MQ1-17H12	Brilliant Violet 421	Effector function
CD40L	Human	TRAP1	Brilliant Violet 480	Co-stimulatory
CD45RO	Human	UCHL1	Brilliant Violet 570	Differentiation
IL-17A	Human	BL168	Brilliant Violet 605	Effector function
Ki-67	Human	B56	Brilliant Violet 650	Proliferation
CD69	Human	FN50	Brilliant Violet 711	Activation
TNF alpha	Human	Mab11	Brilliant Violet 750	Activation
CD27	Human	L128	Brilliant Violet 786	Differentiation

OMIP-059

Identification of Mouse Hematopoietic Stem and Progenitor Cells with Simultaneous Detection of CD45.1/2 and Controllable Green Fluorescent Protein Expression by a Single Staining Panel

Cytometry PART A, Volume 95, Issue 10, 1049-1052 (2019)

Marcus Eich, Andreas Trumpp, Steffen Schmitt

Description: The panel was developed and optimized to identify mouse bone marrow hematopoietic stem cells (HSCs) and five multipotent progenitors (MPPs) along with controllable green fluorescent protein (GFP) expression as well as CD45.1 and CD45.2. HSCs and MPPs can be identified by analyzing the cell surface proteins Sca1, cKit, CD150, CD48, CD34, and CD135. The common myeloid progenitor (CMP), the granulocyte macrophage progenitor (GMP), the megakaryocyte erythrocyte progenitor (MEP), and the common lymphoid progenitor (CLP) are defined by specific expression patterns of Sca1 and cKit plus CD16/32 and CD34, CD135, and CD127. Additionally, the membrane integrity is determined with a life/dead discriminator to differentiate living from dead cells. The staining is performed in freshly isolated living cells without fixation obtained from transgenic mice (SCL‐tTA H2B‐GFP or ISRE‐EGFP) expressing the GFP and the genetic alleles CD45.1 or CD45.2. The analysis of the status of these markers is included in the reported 16 parameter staining panel.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSAria Fusion

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD117	Mouse	2B8	PE	LSK, LS-K
CD34	Mouse	RAM34	Alexa Fluor 700	LT-HSC, ST-HSC, GMP, CMP, MEP
Dead cells	Mouse		Live/Dead Fix Near IR (775)	Viability
CD4	Mouse	Gk1.5	PE-Cy7	Lineage
CD8a	Mouse	53-6.7	PE-Cy7	Lineage
CD11b	Mouse	M1/70	PE-Cy7	Lineage
CD45R (B220)	Mouse	RA3-6B2	PE-Cy7	Lineage
Ly-6G/Ly-6C	Mouse	RB6-8C5	PE-Cy7	Lineage
TER-119	Mouse	TER119	PE-Cy7	Lineage
Ly-6A/Ly-6E	Mouse	D7	Brilliant Violet 421	LSK, LS-K
CD150	Mouse	TC15-12F12.2	Brilliant Violet 785	HSC, MPP
CD48	Mouse	HM48-1	Brilliant Ultraviolet 395	HSC, MPP
Flt-3	Mouse	A2F10	APC	MPP3, MPP4,CLP
cd16/32	Mouse	2.4G2	Brilliant Violet 480	CMP,GMP,MEP
CD127	Mouse	A7R34	Brilliant Violet 650	CLP
CD45.1	Mouse	A20	PE-CF594	internal reporter
CD45.2	Mouse	104	Brilliant Ultraviolet 737	internal reporter

OMIP-058

30‐Parameter Flow Cytometry Panel to Characterize iNKT, NK, Unconventional and Conventional T Cells

Cytometry PART A, Volume 95, Issue 9, 946-951 (2019)

Thomas Liechti, Mario Roederer

Description: This panel enables the in‐depth immunophenotyping of unconventional CD3+ T‐cell populations such as invariant natural killer T cells (iNKT; CD1d:PBS57 tetramer), γδ T cell subsets (TCR Vδ1, TCR Vδ2, TCR Vγ9), and mucosal‐associated invariant T cells (MAIT; CD161, TCR Vα7.2) in combination with conventional CD3+ T cells (CD4, CD8) and natural killer (NK) cell subsets (CD16, CD56) (Table 1). We included markers to assess differentiation stages (CCR7, CD27, CD28, CD45RA, CD57, and CD95) and expression of cytokine receptors (CD122, CD127), activation markers (CD38, HLA‐DR), chemokine receptors (CCR5, CXCR5), the co‐inhibitory molecule PD‐1, and activating and inhibitory NK receptors (CD158, CD244, and CD318). This unique combination of markers enables a thorough phenotypic characterization of these cell subsets (Table 2).

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
HLA-DR	Human	Tu36	PE-Cy5.5	Activation
CD57	Human	NK-1	Brilliant Blue 630-P2	Immune senescence
CD244	Human	25235	Brilliant Blue 660-P2	Co-stimulatory/inhibitory receptor
CD185	Human	RF8B2	Brilliant Blue 790-P	Chemokine receptor
TCR V delta 2	Human	B6	PE-CF594	?d T
CD1d	Human		APC	iNKT
ccr7	Human	150503	Brilliant Ultraviolet 395	Differentiation
PD-1	Human	EH12.1	Brilliant Ultraviolet 661	Inhitibory receptor
CD38	Human	HB7	Brilliant Violet 750	n/a
CD16	Human	3G8	Brilliant Ultraviolet 496	NK
Dead cells	Human		Live/Dead Fix Blue	viability
TCR V delta 1	Human	TS8.2	FITC	?d T
CD127	Human	hIL-7R-M21	Brilliant Blue 700	Cytokine receptor
TCR V gamma 9	Human	B3	PE	?d T
CD161	Human	DX12	PE-Cy5	MAIT
CD314	Human	1D11	PE-Cy7	KIR/NKG2D
CD45RA	Human	HI100	Alexa Fluor 700	Differentiation
CCR5	Human	2D7/CCR5	APC-Cy7	Chemokine receptor
CD56	Human		Brilliant Ultraviolet 563	NK
CD95	Human	Dx2	Brilliant Ultraviolet 737	Differentiation
CD4	Human	SK3	Brilliant Ultraviolet 805	CD4 T
CD122	Human	Mik-b3	Brilliant Violet 421	Cytokine receptor
CD3	Human	UCHT1	Brilliant Violet 510	CD3 T
CD8	Human	RPA-T8	Brilliant Violet 570	CD8 T
CD158	Human	DX27	Brilliant Violet 605	KIR/NKG2D
CD28	Human	CD28.2	Brilliant Violet 650	n/a
TCR V alpha 7.2	Human	3C10	Brilliant Violet 711	n/a
CD27	Human	L128	Brilliant Violet 786	n/a

OMIP-057

Mouse γδ T‐Cell Development Characterized by a 14 Color Flow Cytometry Panel

Cytometry PART A, Volume 95, Issue 7, 726-729 (2010)

Terkild Brink Buus, Mia Hamilton Jee, Niels Ødum

Description: This panel was designed to quantify the distribution of developing γδ T cells within seven development stages related to the programming of distinct effector phenotypes in the murine thymus. Furthermore, the panel was designed to assess the expression of additional surface markers at each development stage within two of the major γδ T‐cell subsets identified by the usage of different V‐segments in their T‐cell receptor (TCR): TCRVγ1.1+ and TCRVγ2+. The panel was developed using thymus from adult C57Bl/6 mice magnetically depleted of CD4 and CD8 expressing cells in order to enrich for the γδ T‐cell population.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD200	Mouse	OX-90	eFluor 660	Population D, E, F
CD24	Mouse	M1/69	Brilliant Violet 510	Population A-F
Dead cells	Mouse		Propidium Iodide	n/a
TCR V gamma 2	Mouse	UC3-10A6	PE-Cy7	?d T-cell subset
CD8	Mouse	53-6.7	Brilliant Ultraviolet 395	Depletion of aß T cells and progenitor
CD4	Mouse	Gk1.5	Brilliant Ultraviolet 737	Depletion of aß T cells and progenitor
CD3	Mouse	145-2C11	Brilliant Violet 786	T cell
CLEC12A	Mouse	5D3/CLEC12A	Brilliant Violet 421	Population A, B
CD25	Mouse	PC61	APC-Cy7	Population A
CD73	Mouse	TY/11.8	Brilliant Violet 605	Population E, F, G
CD117	Mouse	2B8	APC-R700	Population E
TCR delta	Mouse	GL-3	PE-CF594	?d T
TCR V gamma 1.1	Mouse	2.11	FITC	?d T-cell subset

OMIP-056

Evaluation of Human Conventional T Cells, Donor‐Unrestricted T Cells, and NK Cells Including Memory Phenotype by Intracellular Cytokine Staining

Cytometry PART A, Volume 95, Issue 7, 722-725 (2019)

One Dintwe, Shamiska Rohith, Katharine V. Schwedhelm, M. Juliana McElrath, Erica Andersen‐Nissen, Stephen C. De Rosa

Description: A 26‐color staining panel was developed to profile human antigen‐specific T cells in an intracellular cytokine staining (ICS) assay using peptide pools to various antigens of interest. In addition to multiple functional markers, the panel includes differentiation/activation markers and markers to assess γδ, mucosal‐associated invariant T, and NK T cells as well as conventional NK cells. Panel optimization was performed using previously cryopreserved PBMC from healthy adults, and then, expression of key functional markers in the panel was cross‐validated against a validated ICS assay used in the HIV Vaccine Trials Network (HVTN). The panel is currently being used to evaluate the responses to tuberculosis and malaria vaccine candidates in volunteers from different geographic areas.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Blue	Viability
CD45RA	Human	HI100	Brilliant Ultraviolet 496	Differentiation
KLRG1	Human	REA261	PE-Vio615	Differentiation
CD14	Human	MfP9	Brilliant Blue 660-P2	Monocyte
IL-17A	Human	N49-653	Brilliant Ultraviolet 737	Function
CD4	Human	RPA-T4	Brilliant Ultraviolet 805	T cell lineage
IFN-gamma	Human	B27	V450	Function
CD3	Human	UCHT1	Brilliant Ultraviolet 395	T cell lineage
CD40L	Human		PE-Cy7	Function
CD56	Human	CMSSB	PE-Cy5.5	NK,NKT
CD8	Human	RPA-T8	Brilliant Ultraviolet 563	T cell lineage
ccr7	Human	G043H7	Brilliant Violet 785	Differentiation
TNF alpha	Human	Mab11	Brilliant Violet 750	Function
IL-4	Human	MP4-25D2	APC	Function
IL-2	Human	MQ1-17H12	Brilliant Blue 700	Function
Perforin	Human	B-D48	FITC	Function
IL-22	Human	22URTI	PE	Function
CD183	Human	1C6/CXCR3	PE-Cy5	T helper
CCR6	Human		Brilliant Violet 605	T helper
IL-13	Human	JES10-5A2	APC	Function
Granzyme A	Human	CB9	Alexa Fluor 700	Function
TCR V alpha 7.2	Human	REA179	APC-Vio770	MAIT
CD26	Human	M-A261	Brilliant Violet 711	MAIT
HLA-DR	Human	G46-6	Brilliant Ultraviolet 661	Activation
TCR gamma/delta	Human	11F2	Brilliant Violet 480	?d T
CD16	Human	3G8	Brilliant Violet 570	NK,NKT
CD161	Human	DX12	Brilliant Violet 650	MAIT

OMIP-055

Characterization of Human Innate Lymphoid Cells from Neonatal and Peripheral Blood

Cytometry PART A, Volume 95, Issue 4, 427-430 (2019)

Sabrina Bianca Bennstein, Angela Riccarda Manser, Sandra Weinhold, Nadine Scherenschlich, Markus Uhrberg

Description: This OMIP panel was designed to characterize the different subsets of human innate lymphoid cells (ILCs) including ILC1, ILC2, and ILC3 within healthy donors' cord blood (CB) (Table 1) in comparison to peripheral blood (PB). As ILCs represent a rare cell population in blood (0.1–0.5% of lymphocytes), the protocol was designed to rigorously exclude the contaminating cells with a complex lineage‐depleting antibody mixture (13 different antibodies) enabling analysis of highly pure ILC subsets by 8‐color staining.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: Beckman Coulter CytoFLEX

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Green	Viability
CD1a	Human	HI149	FITC	DC
CD14	Human	hCD14	FITC	Monocyte
CD19	Human	HIB19	FITC	B cell
CD20	Human	2H7	FITC	B cell
CD3	Human	UCHT1	FITC	T, NKT
TCR alpha/beta	Human	IP26	FITC	CD3-TCRab+
TCR gamma/delta	Human	B1	FITC	exclude CD3-TCR?d
CD123	Human	6H6	FITC	DC
CD303	Human	201A	FITC	pDC (plasmacytoid DC)
Fc epsilon R1 alpha	Human	AER-37 (CRA-1)	FITC	master cells, basophils
CD235a	Human	HI264	FITC	erythroid cell and erythroid precursor
CD66b	Human	G10F5	FITC	granulocyte
CD34	Human	581	FITC	HSC
CD127	Human	R34.34	PE-Cy5	ILC inclusion marker
CD94	Human	DX22	PE-Cy7	NK
CD56	Human	HCD56	Brilliant Violet 650	NK, ILCs
CD117	Human	104D2	Brilliant Violet 421	ILC3 cell surface receptor
CD294	Human	BM16	PE-Dazzle 594	ILC2 cell surface receptor
CD45	Human	HI30	APC-Cy7	leucocyte
CD161	Human	HP-3G10	Alexa Fluor 700	ILC inclusion marker (especially for tissue)

OMIP-054

Broad Immune Phenotyping of Innate and Adaptive Leukocytes in the Brain, Spleen, and Bone Marrow of an Orthotopic Murine Glioblastoma Model by Mass Cytometry

Cytometry PART A, Volume 95, Issue 4, 422-426 (2019)

Sophie A. Dusoswa, Jan Verhoeff, Juan J. Garcia‐Vallejo

Description: Here we present a 42 parameter panel to characterize myeloid immune cell subsets and T lymphocyte activation status in cryopreserved and barcoded single cell suspensions obtained from brain, spleen, and bone marrow of an orthotopic immunocompetent glioblastoma mouse model in a C57BL/6 background (Table 1). This panel is designed for mass cytometry by time of flight (CyTOF) and combines 34 antibodies against diverse cell surface and intracellular targets together with cisplatin for live/dead discrimination, iridium for cell identification, and six cellular barcodes 1 to enable simultaneous multiplexed acquisition of up to 20 samples (Table 2). The panel is designed for a comprehensive evaluation of the immune system in different organs during murine in vivo studies in the field of glioblastoma immunology, but could also be applied to other disease models in the central nervous system (CNS) with possible systemic involvement, such as brain metastasis arising from other types of cancer, experimental autoimmune encephalomyelitis (EAE), or neurodegenerative disease models. Marker selection was partly based on a combination of previously reported panels studying CNS immune infiltrates 2-7. The selected set of markers captures T lymphocytes (CD8+, CD4+, and regulatory T lymphocytes), dendritic cells (DC), monocytes, macrophages, microglia, tumor cells (when GFP positive), and granulocytes, and contains a set of antibodies to detect cell activation, migratory capacity and immune checkpoints (Table 2). The panel has been optimized with respect to marker selection, antibody clone usage, antibody‐metal pairing, and antibody concentration, and has room for additional markers by filling in a number of free channels as listed in Table 2.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: Fluidigm CyTOF 3 Helios

Panel details

Marker	Species	Clone	Fluorophore	Purpose	Co-expressions groups	Antigen Density

OMIP-053

Identification, Classification, and Isolation of Major FoxP3 Expressing Human CD4+ Treg Subsets

Cytometry PART A, Volume 95, Issue 3, 264-267 (2019)

Johannes Nowatzky, Cristy Stagnar, Olivier Manches

Description: We designed and optimized an inter‐convertible flow cytometry panel for the analysis and sorting of human CD4+ regulatory T cells (Treg) utilizing all of the major, currently accepted marker combinations for Treg identification. The panel is optimized for use with peripheral blood mononuclear cells (PBMC). In addition, the panel allows for the identification, classification, and isolation of activated, antigen‐experienced Treg, and provides estimates of proliferation and suppressive capacity. To this end we created two, easily inter‐convertible sub‐panels: ATREG—which includes intra‐nuclear markers, and BTREG—using surface markers only. Both sub‐panels have been tested on cryopreserved PBMC from healthy donors and those with human autoimmune diseases, as well as on long‐term cultured human Treg and non‐Treg cell lines and clones.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Blue	Viability
HLA-DR	Human	L243	FITC	Activation
CD3	Human	SK7	PerCP-Cy5.5	Lineage
TIGIT	Human	MBSA43	APC	Treg identification
CD127	Human	A019D5	Brilliant Violet 421	Treg (TDP I)
CD4	Human	SK3	Brilliant Violet 510	Lineage
CD8	Human	SK1	Brilliant Violet 650	CD8 exclusion
CD45RA	Human	HI100	Brilliant Violet 711	Treg (TDP II), Treg activation,
CD25	Human	M-A251	PE-Dazzle 594	Treg (TDP I and II)
ccr7	Human	G043H7	PE	Naïve/memory classification
CD38	Human	HB7	Brilliant Ultraviolet 395	Activation (BTREG)
CD226	Human	11A8	PE-Cy7	exclusion of functionally unstable Treg for in vitro expansion, Treg identification (BTREG)
CD39	Human	A1	APC-Fire 750	suppressive capcity and stability under inflammatory condition (BTREG)
FoxP3	Human	236A/E7	APC	Treg (TDP I -III) (ATREG)
Ki-67	Human	B56	Brilliant Ultraviolet 395	Proliferation (ATREG)
Helios	Human	22F6	PE-Cy7	Treg (TDP III) (ATREG)

OMIP-052

An 18‐Color Panel for Measuring Th1, Th2, Th17, and Tfh Responses in Rhesus Macaques

Cytometry PART A, Volume 95, Issue 3, 261-263 (2019)

Mitzi M. Donaldson, Shing‐Fen Kao, Kathryn E. Foulds

Description: This 18‐color panel was designed to measure vaccine‐induced T cell responses in rhesus macaques by intracellular cytokine staining (ICS). It detects seven cytokines, IFN‐γ, IL‐2, IL‐4, IL‐5, IL‐13, IL‐17, and IL‐21, two memory markers, CD45RA and CCR7, and four follicular helper T cell (Tfh) markers, CXCR3, CXCR5, ICOS, and PD‐1 (Figure 1). While this panel was optimized for use on cryopreserved rhesus macaque peripheral blood mononuclear cells (PBMC) (Table 1), it can also be used on rhesus macaque tissue samples and human PBMC and tissue samples.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
IL-4	Rhesus macaque	MP4-25D2	Brilliant Blue 700	cytokine
IL-5	Rhesus macaque	JES1-39D10	Brilliant Blue 515	cytokine
Dead cells	Rhesus macaque		Live/Dead Fix Aqua	Viability
CD3	Rhesus macaque	SP34-2	APC-Cy7	T cell
CD4	Rhesus macaque	S3.5	PE-Cy5.5	T cell
CD8	Rhesus macaque	RPA-T8	Brilliant Violet 570	T cell
CD69	Rhesus macaque	TP1.55.3	ECD	background reduction
IFN-gamma	Rhesus macaque	B27	Alexa Fluor 700	cytokine
IL-2	Rhesus macaque	MQ1-17H12	Brilliant Violet 750	cytokine
IL-13	Rhesus macaque	JES10-5A2	Brilliant Violet 421	cytokine
IL-17	Rhesus macaque	BL168	Brilliant Violet 605	cytokine
IL-21	Rhesus macaque	3A3-N2.1	Alexa Fluor 647	cytokine
CD45RA	Rhesus macaque	5H9	PE-Cy5	memory marker
ccr7	Rhesus macaque	G043H7	Brilliant Violet 650	memory marker
CD183	Rhesus macaque	1C6/CXCR3	Brilliant Violet 711	Tfh
CD185	Rhesus macaque	MU5UBEE	PE	Tfh
PD-1	Rhesus macaque	EH12.2H7	Brilliant Violet 785	Tfh
CD278	Rhesus macaque	C398.4A	PE-Cy7	Tfh

OMIP-051

28‐color flow cytometry panel to characterize B cells and myeloid cells

Cytometry PART A, Volume 95, Issue 2, 105-155 (2019)

Thomas Liechti, Mario Roederer

Description: This 28‐color flow cytometry panel focuses on B cells, dendritic cells, and monocytes and was optimized for cryopreserved peripheral blood mononuclear cells (PBMC). In addition to markers enabling the analysis of monocytes (CD14) and definition of subsets within B cells (CD10, CD19, CD20, CD21, CD27, IgD, IgM) and dendritic cells (CD1c, CD11c, CD123, CD141, HLA‐DR), we included functional markers such as chemokine receptors (CXCR3, CXCR5), surface immunoglobulins (IgA, IgD, IgG, IgM), Fc receptors (CD16, CD23, CD32, CD64), inhibitory molecules (CD73, CD85j), the co‐stimulatory molecule CD40, and cytokine receptors (IL‐21R, BAFF‐R, TACI), which enables in‐depth characterization of B cells, dendritic cells, and monocytes.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD141	Human	1A4	Brilliant Blue 630-P2	DC lineage marker
CD123	Human	7G3	Brilliant Blue 660-P2	DC lineage marker
IgD	Human	IA6-2	Brilliant Blue 790-P	Differentiation B cells
CD21	Human	B-ly4	Brilliant Ultraviolet 496	Differentiation B cells
CD267	Human	1A1-K21-M22	Brilliant Ultraviolet 563	Cytokine receptor
CD185	Human	RF8B2	Brilliant Violet 750	Trafficking
Dead cells	Human		Live/Dead Fix Blue	viability'
CD73	Human	AD2	Brilliant Blue 515	Inhibitory
CD16	Human	3G8	Brilliant Blue 700	Fc receptor
CD32	Human	FUN-2	PE	Fc receptor
CD40	Human	5C3	PE-Dazzle 594	Co-stimulatory
CD85j	Human	GHI/75	PE-Cy5	inhibitory
CD11c	Human	Bu15	PE-Cy5.5	DC lineage marker
CD183	Human	G025H7	PE-Cy7	Trafficking
IgA	Human	polyclonal	APC	Differentiation B cells
CD27	Human	M-T271	APC-R700	Differentiation B cells
CD19	Human	SJ25C1	APC-H7	Lineage B cell
CD1c	Human		Brilliant Ultraviolet 395	DC lineage marker
HLA-DR	Human	G46-6	Brilliant Ultraviolet 661	DC lineage marker
IgG	Human		Brilliant Ultraviolet 737	Differentiation B cells
CD20	Human	2H7	Brilliant Ultraviolet 805	Lineage B cell
IL-21 R	Human	2G1-K12	Brilliant Violet 421	Cytokine receptor
CD14	Human		Brilliant Violet 510	Lineage monocyte
IgM	Human	MHM-88	Brilliant Violet 570	Differentiation B cells
CD268	Human	11C1	Brilliant Violet 605	Cytokine receptor
CD10	Human	HI10a	Brilliant Violet 650	Differentiation B cells
CD23	Human	M-L233	Brilliant Violet 711	Differentiation B cells
CD64	Human	10.1	Brilliant Violet 786	Fc receptor

OMIP-050

A 28‐color/30‐parameter Fluorescence Flow Cytometry Panel to Enumerate and Characterize Cells Expressing a Wide Array of Immune Checkpoint Molecules

Cytometry PART A, Volume 93, Issue 11, 1094-1096 (2018)

Leonard Nettey, Amber J. Giles, Pratip K. Chattopadhyay

Description: This 28‐color/30‐parameter panel catalogs, enumerates and characterizes cells expressing molecules that regulate T‐cell responses (“checkpoint” molecules). The primary purpose is to measure combinatorial expression of PD‐1, CTLA‐4, TIM‐3, CD244/2B4, TIGIT, BTLA, CD137/4‐1BB, GITR, OX40, CD27, and CD278/ICOS, many of which are targets of the immunotherapy drugs in preclinical or clinical development. This panel is optimized for cryopreserved healthy human peripheral blood mononuclear cells (PBMCs) and cryopreserved mononuclear cells isolated from tumor specimens (tissue MNCs). CD45 is used to distinguish leukocytes from stromal cells and debris often present in tissue digests. CD3, CD4, and CD8 identify T‐cells, whose differentiation status is then assessed with CD45RO, CD95, CCR7, CD27, CD57, and CD28. CD25, HLA‐DR, and CD69 mark activated T‐cells. CD69 is also used to identify tissue‐resident lymphocytes in combination with CD103. CXCR3 and CXCR6 mediate lymphocyte trafficking toward chemokines often found in the tumor microenvironment. Regulatory T‐cells are defined here by antibodies against CD25 and CD127. In sum, this panel comprehensively characterizes cells expressing checkpoint markers.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD186	Human	13B 1E5	Brilliant Blue 515	Trafficking
CD137	Human	4B4-1	Brilliant Blue 630-P2	co-stimulatory
TIGIT	Human	MBSA43	Brilliant Blue 660-P2	co-inhibitory
CD57	Human	NK-1	Brilliant Blue 790-P	Differentiation
CD69	Human	FN50	Brilliant Violet 750	Activation
ccr7	Human	150503	Brilliant Ultraviolet 395	Lineage
CD366	Human	7D3	Brilliant Ultraviolet 661	co-inhibitory
CD244	Human	C1.7	PE-Cy5.5	co-inhibitory
CD27	Human	O323	PE	Differentiation
Dead cells	Human		Live/Dead Fix Blue	Viability
CD28	Human	L293	Brilliant Blue 700	Differentiation
CD278	Human	DX29	Brilliant Violet 421	co-stimulatory
CD95	Human	Dx2	Brilliant Violet 480	Differentiation
CD8	Human	RPA-T8	Brilliant Violet 570	Lineage
CD103	Human	Ber-ACT8	Brilliant Violet 605	Differentiation
CD183	Human	G025H7	Brilliant Violet 650	Trafficking
CD134	Human	L106	Brilliant Violet 711	co-stimulatory
PD-1	Human	EH12.2H7	Brilliant Violet 785	co-inhibitory
CD3	Human	UCHT1	Brilliant Ultraviolet 496	Lineage
CD25	Human	2A3	Brilliant Ultraviolet 563	Differentiation
CD4	Human	SK3	Brilliant Ultraviolet 737	Lineage
CD45	Human	HI30	Brilliant Ultraviolet 805	Lineage
CD45RO	Human	UCHL1	APC	Differentiation
HLA-DR	Human	G46-6	APC-R700	Activation
CD357	Human	108-17	APC-Fire 750	co-stimulatory
CD272	Human	J168-540	PE-CF594	co-inhibitory
CD127	Human	A019D5	PE-Cy5	Differentiation
CTLA-4	Human	L3D10	PE-Cy7	co-inhibitory

OMIP-049

Analysis of Human Myelopoiesis and Myeloid Neoplasms

Cytometry PART A, Volume 93, Issue 10, 982-986 (2018)

Genyuan Zhu Jason Brayer, Eric Padron, James J. Mulé, Adam W. Mailloux

Description: Cells undergoing myelopoiesis in both bone marrow and the mature myeloid compartments in the blood represent a broad range of phenotypes in different stages of maturation and activation. Neoplasms that arise from this lineage are often heterogeneous and frequently display unique characteristics and outcomes depending on the originating subset or degree of differentiation 1, 2. This is particularly true for myeloid neoplasms (MN) which rely on multiple, separate flow cytometry panels for differential diagnosis 3, 4. While standard practice, this approach has inherent disadvantages which include the inability to observe co‐expression patterns between markers contained in separate panels and increased time and labor associated with multiple flow cytometry assays. Use of a single, high‐order panel that is able to differentiate multiple hematopoietic lineages, or distinguish different MNs would aid this effort and provide the opportunity to further characterize disease subsets. This panel was designed to detect cell surface markers associated with hematopoiesis with a special emphasis on the progression of myelopoiesis from hematopoietic progenitors. Because the study of hematopoietic disease requires a broad combination of early hematopoietic and mature myeloid markers 5, this panel is also appropriate for the general analysis of myelopoiesis, granulopoiesis, erythropoiesis, and megakaryocytopoiesis, in any human cell source that contains mature myeloid cells, myeloid progenitors, or hematopoietic progenitors (Table 1).

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Aqua	Viability
CD138	Human	MI15	APC-Cy7	Plasma cell
CD19	Human	HIB19	Brilliant Violet 510	exclusion
CD3	Human	SK7	Brilliant Violet 510	exclusion
CD45	Human	HI30	Brilliant Ultraviolet 805	Leukocyte
CD117	Human	104D2	Brilliant Blue 515	Progenitor
CD11b	Human	ICRF44	Brilliant Violet 650	Myeloid Lineage
CD11c	Human	3.9	PE-CF594	DC
CD14	Human	M5E2	Brilliant Violet 786	Monocyte
CD16	Human	3G8	Brilliant Ultraviolet 496	FC receptor
CD33	Human	WM53	Brilliant Violet 421	Myeloid Lineage
CD34	Human	581	PE-Cy7	Progenitor
CD36	Human	CB38	PerCP-Cy5.5	M2
CD64	Human	10.1	Brilliant Ultraviolet 737	Activation, M1
HLA-DR	Human	G46-6	Brilliant Ultraviolet 395	M1
CD15	Human	W6D3	Alexa Fluor 700	Myeloid Lineage
CD163	Human	GHI/61	Brilliant Violet 605	Macrophage
CD32	Human	FUN-2	APC	FC receptor
cd41a	Human	HIP8	PE	Megakaryocyte
CD71	Human	M-A712	Brilliant Violet 711	Erythroid marker

OMIP-048

Quantification of calcium sensors and channels expression in lymphocyte subsets by mass cytometry

Cytometry PART A, Volume 93, Issue 7, 681-684 (2018)

Agnieszka Jaracz‐Ros, Patrice Hémon, Roman Krzysiek, Françoise Bachelerie, Géraldine Schlecht‐Louf, Hélène Gary‐Gouy

Description: The present mass cytometry panel 1, 2 was designed to simultaneously assess the ex vivo expression of 2 calcium (Ca2+)‐sensors and 4 Ca2+‐channels, the key components of the T cells “Ca2+‐toolkit,” in murine conventional (Tconv) and Foxp3+ regulatory (Treg) T‐cell subsets, including both naive and activated populations (Table 1), after a validation step of the selected antibodies (Abs, listed in Table 2) by flow cytometry.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa & Fluidigm CyTOF 1

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD3 epsilon	Mouse	145-2C11	Purified	Lineage
CD19	Mouse	6D5	Purified	Lineage
DNA	All Species		Purified	Nucleated cells
CD45R	Mouse	RA3-6B2	Purified	Lineage
FoxP3	Mouse	FJK-16s	Purified	Lineage
CD25	Mouse	PC61	Purified	Activation
ORAI2	Mouse		Purified	Ca2+-channel
STIM2	Mouse		Purified	Ca2+-sensor
DNA	All Species		Purified	Viability
CD4	Mouse	RM4-5	Purified	Lineage
CD44	Mouse	IM7	Purified	Activation
CD5	Mouse	53-7.3	Purified	Lineage
CD62L	Mouse	Mel-14	Purified	Activation
CD8a	Mouse	53-6.7	Purified	Lineage
VDAC1	Mouse	20B12AF2	Purified	Ca2+-sensor/channel
STIM1	Mouse	CDN3H4	Purified	Ca2+-channel
ORAI1	Mouse	3F6H5	Purified	Ca2+-channel
ORAI3	Mouse		Purified	Ca2+-channel
Ki-67	Mouse	B56	Purified	Proliferation
CD45	Mouse	30-F11	Purified	Lineage

OMIP-047

High‐Dimensional phenotypic characterization of B cells

Cytometry PART A, Volume 93, Issue 6, 592-596 (2018)

Thomas Liechti, Huldrych F. Günthard, Alexandra Trkola

Description: This 16‐color, 18‐parameter panel was designed to allow a detailed dissection of human B cell subsets and their phenotype in peripheral blood mononuclear cells (PBMC) in healthy donors and in the context of chronic viral diseases such as Human Immunodeficiency Virus 1 (HIV‐1) infection. The panel encompasses a range of backbone markers for the accurate definition of common B cell subsets with a focus on memory B cells and a unique collection of phenotypic markers (chemokine receptors, cytokine receptor, B cell receptor isotypes, and proliferation marker) not combined in multicolor flow cytometry B cell phenotyping thus far. This new panel allows highly detailed phenotypic and functional investigations of B cell subsets. The panel was validated using cryopreserved PBMC from healthy and HIV‐1 infected donors allowing the retrospective analysis of clinical samples (Table 1).

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
IgG1	Human	HP6001	PE	IgG1 calss-switching B cell
IgG3	Human		FITC	IgG3 calss-switching B cell
CXCR4	Human	12G5	PE-Cy5.5	Migration pattern
IgD	Human	IA6-2	PE-Cy5	B cell subset
CD3	Human	SK7	APC-Cy7	Dump
CD14	Human	hCD14	APC-Cy7	Dump
CD16	Human	3G8	APC-Cy7	Dump
Dead cells	Human		Live/Dead Fix Near IR (775)	Viability
CD19	Human	SJ25C1	Brilliant Violet 786	Lineage
CD10	Human	HI10a	Brilliant Violet 650	B cell subset
CD38	Human	HIT2	Alexa Fluor 700	B cell subset
CD21	Human	B-ly4	Brilliant Violet 711	B cell subset/Exhaustion
CD27	Human	M-T271	PE-CF594	Differentitation, memory
IgA	Human		APC	IgA calss-switching B cell
ccr7	Human	G043H7	Brilliant Violet 605	Migration pattern
CD183	Human	G025H7	PE-Cy7	Migration pattern
CD185	Human	RF8B2	Brilliant Violet 510	Migration pattern
IL-21 R	Human	2G1-K12	Brilliant Violet 421	Cytokine receptor
Ki-67	Human	20Raj1	PerCP-eFluor 710	Proliferation marker

OMIP-046

Characterization of invariant T cell subset activation in humans

Cytometry PART A, Volume 93, Issue 5, 499-503 (2018)

Kerri G. Lal, Edwin Leeansyah, Johan K. Sandberg, Michael A. Eller

Description: This panel was developed to characterize the two most commonly studied invariant T cell subsets: mucosal‐associated invariant T (MAIT) cells and invariant natural killer T (iNKT) cells in humans and measure their expression of exhaustion and activation markers. The panel was optimized and designed for application in cryopreserved peripheral blood mononuclear cells (PBMC) (Table 1). Designed to be used to measure frequency and phenotype of invariant T cell subtypes in acute HIV‐1 infection, optimization was performed on chronically HIV‐1 infected and uninfected donors and can be applied to a variety of human disease cohorts.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2 SORP

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD8	Human	SK1	PerCP-Cy5.5	T cell lineage
TIGIT	Human	MBSA43	PE-Cy7	Exhaustion
Dead cells	Human		Live/Dead Fix Aqua	Viability
TCR V alpha 24	Human	C15	FITC	iNKT
CD57	Human	hCD57	Pacific Blue	Activation
CD161	Human	HP-3G10	Brilliant Violet 605	MAIT
HLA-DR	Human	L243	Brilliant Violet 650	Activation
CD38	Human	HIT2	Brilliant Violet 711	Activation
PD-1	Human	EH12.1	Brilliant Violet 786	Exhaustion
TCR V alpha 7.2	Human	3C10	APC	MAIT
CD14	Human	M5E2	Alexa Fluor 700	Exclusion
CD19	Human		Alexa Fluor 700	Exclusion
CD4	Human	SK3	APC-H7	T cell lineage
TCR V beta 11	Human	C21	PE	iNKT
CD3	Human	S4.1	PE-Texas Red	T cell lineage

OMIP-045

Characterizing human head and neck tumors and cancer cell lines with mass cytometry

Cytometry PART A, Volume 93, Issue 4, 406-410 (2018)

Tess M. Brodie, Vinko Tosevski, Michaela Medová

Description: A 42‐parameter panel (34 antibody targets, cisplatin for live/dead, iridium for cell identification, and 6 barcodes) was developed for the characterization of cell types and signaling pathways present in tumor tissues or cell lines originating from primary, metastatic and recurrent head and neck squamous cell carcinoma (HNSCC), the most prevalent malignant tumor of the head and neck region and the 6th most common form of non‐skin cancer worldwide 1 (Table 1). Importantly, due to overlap in targets and markers across diverse tumor entities [e.g., HER2 amplification is present in both breast and gastric tumors 2, 3], the described panel may be appropriate for use in preclinical models of additional distinct solid as well as hematological malignancies. This panel has been successfully tested on fresh, formalin‐fixed, and frozen cells originating from cell lines derived from primary and metastatic/recurrent tumors and from patients' tumor tissue.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: Fluidigm CyTOF 2.1 Helios

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD29	Human	TS2/16	Purified	Metastasis
CD45	Human	HI30	Purified	Leukocytes
CD31	Human	390	Purified	Endothelial cells
CD10	Human	HI10a	Purified	Cancer associated fibroblasts
Vimentin	Human	D21H3	Purified	Mesenchymal cells
CD68	Human	Y1/82A	Purified	Macrophages and DCs
CD24	Human	ML5	Purified	B cells
CD324	Human	24E11	Purified	Epithelial cells
CD274	Human	29E.2A3	Purified	Tolerance promotion
CD28	Human	CD28.2	Purified	T cell activation
Ki-67	Human	B56	Purified	Proliferation
p53	Human	261352	Purified	Tumor suppressor protein
PARP, cleaved	Human	F21-852	Purified	Repair of single stranded DNA
ERK1/2 (pT202/pY204)	Human	D13.14.4E	Purified	Proliferation
EGFR (pY845)	Human	D7A5	Purified	Proliferation
HER2/ErbB2	Human	29D8	Purified	Proliferation
Akt (pS473)	Human	D9E	Purified	Survival
EGF Receptor	Human		Purified	Proliferation
Hes1	Human	4H1HES1	Purified	Transcription suppressor
c-Met	Human	eBioclone 97	Purified	Proliferation
S6 (pS235/pS236)	Human	N7-548	Purified	Increased translation
CD166	Human	3A6	Purified	Associated with cancer stem cells
CD44	Human		Purified	Associated with cancer stem cells
PD-1	Human	EH12.2H7	Purified	Tolerance promotion
MDR1	Human	UIC2	Purified	Drug resistance
CD3	Human	SK7	Purified	T cells
Met (pY1234/pY1235)	Human		Purified	Proliferation
DDR2	Human	290804	Purified	Proliferation
TRAF3	Human	B1-6	Purified	Associated with HNSCC
YAP1	Human	867711	Purified	Proliferation
EphA2	Human	SHM16	Purified	Migration and proliferation
MMP-9	Human	4H3	Purified	Metastasis
TIMP-3	Human	277128	Purified	Metastasis inhibitor
ALDH1A1	Human	703410	Purified	Associated with cancer stem cells

OMIP-044

28‐color immunophenotyping of the human dendritic cell compartment

Cytometry PART A, Volume 93, Issue 4, 402-405 (2018)

Florian Mair, Martin Prlic

Description: This 28‐color panel has been developed for an extensive phenotyping of antigen‐presenting cells (APCs) in human blood and tissue samples (Table 1). 15 markers associated with changes in dendritic cell (DC) function were selected based on the current literature. Five additional markers were used to pregate on canonical DC subpopulations, namely CD141+ cross‐presenting conventional DC1 (cDC1), CD1c+ conventional DC2 (cDC2), CD141‐ CD1c‐ cDCs as well as CD123+ plasmacytoid DCs (pDC). Furthermore, seven lineage markers were included for parallel enumeration of CD14+ monocytes, B cells, NK cells as well as CD4+ and CD8+ αβ T cells with basic phenotyping of their differentiation status. The panel has been tested on cryopreserved peripheral blood mononuclear cells (PBMCs) and allows further inclusion of DC antigens at the cost of pan‐phenotyping markers.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSymphony A5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Blue	Viability
CD272	Human	J168-540	Brilliant Blue 630-P2	DC phenotype
CD8	Human	RPA-T8	Brilliant Blue 660-P2	CD8 T cell
CD38	Human	HIT2	Brilliant Blue 790-P	DC phenotype
CD40	Human	5C3	Brilliant Ultraviolet 395	DC phenotype
CD16	Human	3G8	Brilliant Ultraviolet 496	DC phenotype, monocyte phenotype
CD56	Human	NCAM16.2	Brilliant Ultraviolet 563	NK
CD3	Human	UCHT1	Brilliant Ultraviolet 661	T cell
CD86	Human	FUN-1	Brilliant Ultraviolet 737	DC phenotype
CD45	Human	HI30	Brilliant Ultraviolet 805	hematopoitic cell
CX3CR1	Human	2A9-1	Brilliant Violet 421	DC phenotype
CD85k	Human	ZM3.8	Brilliant Violet 480	DC phenotype
CD45RA	Human	HI100	Brilliant Violet 570	DC, T cell
CD141	Human	1A4	Brilliant Violet 605	DC subset
CD172a	Human	SE5A5	Brilliant Violet 650	DC phenotype
CD14	Human	MfP9	Brilliant Violet 711	monocyte
CD11b	Human	ICRF44	Brilliant Violet 750	DC phenotype
CD123	Human	7G3	Brilliant Violet 786	pDC (plasmacytoid DC)
CD26	Human	M-A261	FITC	DC subset
CD32	Human	FLI8.26	Brilliant Blue 700	DC phenotype
ccr7	Human	G043H7	PE	DC phenotype, T cell phenotype
CD163	Human	GHI/61	PE-CF594	DC phenotype
CD80	Human	L307.4	PE-Cy5	DC phenotype
CD19	Human	SJ25-C1	PE-Cy5.5	B cell
CD4	Human	SK3	PE-Cy7	CD4 T cell
CD1c	Human	F10/21A3	Alexa Fluor 647	DC subset
CD11c	Human	B-ly6	Alexa Fluor 700	DC lineage marker
HLA-DR	Human	G46-6	APC-H7	DC lineage marker

OMIP-043

Identification of human antibody secreting cell subsets

Cytometry PART A, Volume 93, Issue 2, 190–193 (2018)

Jeffrey Carrell, Christopher J. Groves

Description: This panel was optimized primarily to determine the frequency and immunophenotype of antibody secreting cells (ASC), historically called plasma B cells [PCs, reviewed in Refs. ( 1-3)]. The panel can also be used to determine the frequency and phenotype of other B cell subsets including memory B cells and naïve B cells, which occur in various anatomic niches, but particularly in the circulation (Table 1). The panel has been tested on various human tissues from healthy subjects, and has been shown to be applicable to fresh and cryopreserved peripheral blood, spleen, bone marrow, and tonsil cells; we have observed active antibody secretion from thawed cells. We continue to apply the panel to a variety of tissues and donors to build on our understanding of humoral immunity and the cells that contribute to long‐lasting vaccine responses. Analysis of rare cells can be difficult, and our aim is to encourage better data in the analysis of ASC.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD SORP Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Blue	viability
CD20	Human	2H7	Brilliant Violet 421	B cell differentiation
CD27	Human		Brilliant Violet 785	B cell differentiation
CD38	Human	HB7	Brilliant Ultraviolet 395	Primary identificaiton of ASCs
CD19	Human	SJ25C1	APC	B cell differentiation
IgD	Human	IA6-2	PerCP-Cy5.5	B cell maturation
CD3	Human	UCHT1	Brilliant Violet 510	Dump/exclusion
CD14	Human	MfP9	Brilliant Violet 510	Dump/exclusion
CD15	Human	W6D3	Brilliant Violet 510	Dump/exclusion
CD193	Human		Brilliant Violet 510	Dump/exclusion
IgM	Human	G20-127	Brilliant Violet 605	surface Ig
IgM	Human	G18-145	FITC	Cytoplasmic Ig
IgG	Human		Brilliant Violet 605	surface Ig
IgG	Human	G20-127	FITC	Cytoplasmic Ig
IgA	Human	PAb	FITC	Cytoplasmic Ig
IgE	Human		PE-Cy7	Cytoplasmic Ig
Ki-67	Human	N/A	PE	Proliferation

OMIP-042

21‐color flow cytometry to comprehensively immunophenotype major lymphocyte and myeloid subsets in human peripheral blood

Cytometry PART A, Volume 93, Issue 2, 186–189 (2018)

Karl W. Staser, William Eades, Jaebok Choi, Darja Karpova, John F. DiPersio

Description: This 21-color flow cytometry-based OMIP [1] enables simultaneous quantification of monocytes, basophils, granulocytes, dendritic cells, natural killer cells, B cells, and all well-defined T and T helper cell subsets in the human peripheral blood (Table 1). This panel captures the major phenotypes described in the NIH Human Immunology Project [2, 3] with additional markers for deep T cell analysis [4]. We specifically designed this panel for analysis of peripheral blood from patients involved in our clinical trials of novel agents for the treatment of graft versus host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (alloHSCT). We have optimized this panel for the analysis of 1 × 106 fresh or previously frozen peripheral blood mononuclear cells (PBMCs).

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: Bio-Rad Ze5

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CCR10	Human	314305	APC	Th subset
Dead cells	Human		Live/Dead Fix Blue	viability
CD14	Human	MfP9	Brilliant Ultraviolet 395	monocyte
CD16	Human	3G8	Brilliant Ultraviolet 496	monocyte
HLA-DR	Human	G46-6	Brilliant Ultraviolet 661	DC
CD56	Human	NCAM16.2	Brilliant Ultraviolet 737	NK
CD38	Human	HIT2	Brilliant Violet 421	actvation
CD20	Human	L27	V450	B cell
CD4	Human	SK3	Brilliant Violet 510	CD4 T cell
CD194	Human	L291H4	Brilliant Violet 605	Th subset
CD8	Human	RPA-T8	Brilliant Violet 650	CD8 T cell
CD25	Human	2A3	Brilliant Violet 711	Treg
CCR6	Human		Brilliant Violet 785	Th subset
CD3	Human	UCHT1	Alexa Fluor 488	T cell
CD45RA	Human	HI100	PerCP-Cy5.5	naïve/memory
CD183	Human	1C6	PE	Th subset
ccr7	Human	150503	PE-CF594	central/effector
CD11c	Human		PE-Cy5	mDC
CD185	Human	RF8B2	PE-Cy7	Th subset
CD123	Human	32703	Alexa Fluor 700	pDC (plasmacytoid DC)
CD127	Human	RDR5	APC-eFluor 780	Treg

OMIP-041

Optimized multicolor immunofluorescence panel rat microglial staining protocol

Cytometry PART A, Volume 93, Issue 2, 182–185 (2017)

Naama E. Toledano Furman, Karthik S. Prabhakara, Supinder Bedi, Charles S. Cox Jr, Scott D. Olson

Description: The common usage of animal models in a variety of preclinical studies is supported by appropriate species‐specific antibodies to be utilized in immunohistochemistry (IHC), western blotting, and flow cytometry (FC) assays. Other than the technical advantages (sophisticated surgical manipulations due to their size), modest cost (relative to larger animals), and the standardized results, the similarities in metabolic activity and physiology of neurological disorders to humans make rats appropriate for neurological disease or disorders models 1, 2. However, rat‐based assays are not as comprehensive or standardized as mouse or human based assays are, partially because there is a shortage in rat‐specific antibodies. Rat‐specific antibodies are now becoming commercially available, which allows us to set standardize criteria for rat origin cells of interest. As our research focus is in neurotherapy, we are interested specifically in microglial cells, which are the innate immune cells in the brain 3 and spinal cord 4. Microglial cells play a critical role in traumatic brain injuries (TBI) and spinal cord injuries (SCI), and their presence, activation, and effect are highly investigated in those models 4-6. Microglial characterization via FC would save many hours of work as a substitute for IHC analysis, yield unbiased statistics, and overall help research move at a faster pace 7. Here we present multicolor phenotyping panels for assessing microglia derived from rat brain or spinal cord for their activation states, polarization, and number (see Table 1). The microglial cells used are immediately isolated from fresh brain or spinal cord tissues, using a Neural Tissue Dissociation kit, followed by myelin removal and purification using anti‐rat CD11b/c microbeads.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
P2RY12	Rat	polyclonal	Brilliant Violet 421	PE
CD163	Rat	ED2	PerCP-Cy5.5	V510
CD200 R	Rat	OX-102	PE	Alexa Fluor 647
Dead cells	Rat	N/A	Ghost Dye Violet 510	APC-Cy7
CD45	Rat	OX-1	APC-Cy7	PE-Cy7
CD11b/c	Rat	OX-42	PE-Cy7	BV421
CD32	Rat	D34-485	PE	APC
CD86	Rat		APC	PE
RT1B	Rat	OX-6	Alexa Fluor 647	PerCP Cy5.5

OMIP-040

Optimized gating of human prostate cellular subpopulations

Cytometry PART A, Volume 91, Issue 12, 1147–1149 (2017)

Gervaise H. Henry, Nicolas Loof, Douglas W. Strand

Description: This panel was optimized to quantify the relative frequency of the major cell types present in the human prostate in addition to their sorting for downstream applications. Tissue resident white blood cells (referred to here as leukocytes) are identified by CD45, epithelia are identified by CD326, and stroma are double negative with those markers. Epithelia can be further segregated into basal, luminal, and “other” populations using CD26 and CD271. Fibromuscular stroma can be identified by removing CD31‐positive endothelia from the stroma. This panel can also serve as a backbone for the addition of markers to interrogate subpopulations within these major cell types. The panel has been validated on freshly digested and cryopreserved human prostate cells collected from young organ donors, BPH patients, and prostate cancer patients. Other tissue types have not been tested. Other basal cell markers including CD49f, podoplanin, and CD104 are tested and compared with CD271.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSAria Fusion SORP

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD45	Human	H130	PerCP-Cy5.5	leukocyte
Dead cells	Human		Ghost Dye Red 780	viability
CD326	Human	EBA-1	Brilliant Blue 515	epithelia
NGF Receptor p75	Human	ME20.4	PE	basal epithelia
CD26	Human	BA5b	APC	luminal epithelia
CD31	Human	WM59	Brilliant Violet 421	endothelia

OMIP-039

Detection and analysis of human adaptive NKG2C+ natural killer cells

Cytometry PART A, Volume 91, Issue 10, 997–1000 (2017)

Quirin Hammer, Chiara Romagnani

Description: The present panel was optimized for the detection of adaptive natural killer (NK)‐cell populations in healthy human donors and offers versatility to investigate their biology including receptor usage, activation requirements, or signaling adaptor and transcription factor expression. It was established on cryopreserved PBMC and yields similar results with freshly isolated PBMC. Additional tissue samples have not been tested (Table 1).

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD159c	Human	REA205	PE	Adaptive NK
CD159a (NKG2a)	Human	REA110	PE-Vio770	Conventional NK
Dead cells	Human		Zombie Aqua	viability
CD14	Human	M5E2	Brilliant Violet 510	exclusion
CD19	Human	HIB19	Brilliant Violet 510	exclusion
CD3	Human	UCHT1	PE-Cy5	exclusion
CD56	Human	HCD56	PE-Dazzle 594	NK cells
CD337	Human	AF29-4D12	eFluor 450	Conventional NK
CD328	Human	REA214	APC-Vio770	Conventional NK
CD85j	Human	HP-F1	APC	Adaptive NK
CD57	Human	TBO1	Purified	Adaptive NK
IgM	Human	RMM-1	Brilliant Violet 605	secondary reagent for CD57
CD2	Human	RPA-2.10	PerCP-Cy5.5	Adaptive NK
CD7	Human	M-T701	Brilliant Violet 786	Conventional NK

OMIP-038

Innate immune assessment with a 14 color flow cytometry panel

Cytometry PART A, Volume 91, Issue 10, 966–968 (2017)

Kinga K. Smolen, Bing Cai, Tobias R. Kollmann

Description: The purpose of the panel presented here is to assess the innate immune response in whole blood after Toll‐like receptor (TLR) ligand or cytokine stimulation, at the single‐cell level. In addition to the identification of many of the innate and innate‐adaptive interface immune cell subsets, this panel also enables the detection of key intracellular cytokines involved in the immune response.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
IL-12	Human	C8.6	eFluor 450	Function
IL-6	Human	MQ2-13A5	PerCP-eFluor 710	Function
TCR gamma/delta	Human	B1.1	FITC	?d T cell
CD123	Human	6H6	PE-Cy7	pDC (plasmacytoid DC)
CD66	Human	ASL-32	Biotin	Neutrophil
Streptavidin	Human		Brilliant Violet 786	secondary reagent to CD66
Dead cells	Human		eFluor 780 Fix Viability	Live/Dead
HLA-DR	Human	LN3	eFluor 605NC	B cell, monocyte
CD11c	Human	S-HCL-3	APC	cDC
TNF alpha	Human	Mab11	Alexa Fluor 700	Function
CD3	Human	UCHT1	PE-CF594	T lineage
IFN-alpha	Human	7N4-1	PE	Function
IFN-gamma	Human	4S.B3	Brilliant Violet 711	Function
CD16	Human	3G8	Brilliant Violet 650	NK, NKT, monocyte
CD14	Human	M5E2	V500	Monocytes

OMIP-037

16-color panel to measure inhibitory receptor signatures from multiple human immune cell subsets

Cytometry PART A, Volume 91, 175–179 (2016)

Anna C. Belkina, Jennifer E. Snyder-Cappione

Description: The panel was developed to determine the combinational inhibitory receptor expression (“IR signatures”) of CD4+ T cells, CD8+ T cells, Natural Killer (NK) cells, invariant Natural Killer T (iNKT) cells, and gamma delta (γδ) T cells from individual human samples. The inhibitory receptors measured are PD-1, TIM-3, CD160, LAG-3, and TIGIT. The activation marker CD137 (4-1BB) is also included in the panel, as is IL-7Rα (CD127). This panel works well with cryopreserved PBMC from healthy and HIV-infected individuals well as fresh tumor specimens. For optimum performance of this panel with digested tumor specimens, the pan-lymphocyte marker CD45 is included (swapped for LAG-3). Other tissues/sample types have not yet been evaluated. Also, a modification of this panel has been optimized that includes CD45RO and CD25 in place of CD160 and CD137.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSAria II SORP

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD160	Human	BY55	Alexa Fluor 488	Exhaustion
TCR V alpha 24	Human	C15	PE	iNKT lineage
CD1d	Human		APC	iNKT lineage
CD16	Human	3G8	FITC	NK cell lineage, subsets
Dead cells	Human		Zombie NIR	Viability
TCR gamma/delta	Human	B1	Brilliant Ultraviolet 395	?d T cell
CD127	Human	hIL-7R-M21	Brilliant Ultraviolet 737	Exhaustion, Treg
CD8	Human	SK1	Brilliant Ultraviolet 805	Lineage
PD-1	Human	EH12.2H7	Brilliant Violet 421	Exhaustion
CD3	Human	OKT3	Brilliant Violet 510	T cell lineage
CD16	Human	3G8	Brilliant Violet 605	NK lineage/subsets
CD137	Human	4B4-1	Brilliant Violet 650	Acivation
CD56	Human	NCAM16.2	Brilliant Violet 786	NK lineage/subsets
Lag-3	Human	3DS223H	PE-eFluor 610	Exhaustion
TIGIT	Human	MBSA43	PerCP-eFluor 710	Exhaustion
TIM-3	Human	F38-2E2	PE-Cy7	Exhaustion
CD4	Human	RPA-T4	Alexa Fluor 700	Th lineage
CD14	Human	hCD14	APC-Cy7	monocyte exclusion
CD19	Human	HIB19	APC-Cy7	B cell exclusion
CD45	Human	HI30	PE-Dazzle 594	hematopoitic cell ineage
CD25	Human	BC96	Brilliant Violet 605	Treg, T cell activation
CD45RO	Human	UCHL1	Brilliant Violet 650	naïve/memory

OMIP-036

Co-inhibitory receptor (immune checkpoint) expression analysis in human T cell subsets

Cytometry PART A, Volume 89, Issue 10, 889–892 (2016)

Zachary R. Healy, David M. Murdoch

Description: This panel was optimized to quantify inhibitory receptor expression on CD4 and CD8 T cells from differentiation and activation subsets. Six inhibitory (i.e., immune checkpoint) receptors (PD-1, TIM-3, LAG-3, CD160, BTLA, CTLA-4) were chosen based upon previously published observations suggesting their role in modulating CD4 and CD8 T cell activation in response to persistent antigen exposure [1-3]. Furthermore, given the important observations that inhibitory receptor expression varies by differentiation and prior antigen experience, markers of T cell differentiation status and prior antigen experience (CCR7, CD45RA, CD28, CD127, KLRG1) were also included [4-6]. This panel was developed and optimized for use in cryopreserved human peripheral blood mononuclear cells (PBMCs), although it has also been applied in fresh PBMCs as well as other bodily fluids (e.g., malignant ascites) (Table 1).

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Lag-3	Human	3DS223H	PE-Cy7	Inhibitory Receptors
Dead cells	Human		Zombie Aqua	Viability
CD14	Human	M5E2	Brilliant Violet 510	Dump/exclusion
CD19	Human	HIB19	Brilliant Violet 510	Dump/exclusion
CD3	Human	SK7	Brilliant Ultraviolet 395	Phenotyping
CD4	Human	SK3	Brilliant Ultraviolet 496	Phenotyping
CD8	Human	SK1	Brilliant Ultraviolet 805	Phenotyping
ccr7	Human	G043H7	Brilliant Violet 785	Maturation/Activation
CD45RA	Human	HI100	Brilliant Blue 515	Maturation/Activation
CD28	Human	CD28.2	APC-H7	Maturation/Activation
CD127	Human	A019D5	Brilliant Violet 650	Maturation/Activation
KLRG1	Human	SA231A2	Alexa Fluor 647	Maturation/Activation
PD-1	Human	EH12.2H7	Brilliant Violet 711	Inhibitory Receptors
TIM-3	Human	F38-2E2	Brilliant Violet 605	Inhibitory Receptors
CD160	Human	BY55	PerCP-Cy5.5	Inhibitory Receptors
CD272	Human	MIH26	Brilliant Violet 421	Inhibitory Receptors
CTLA-4	Human	BNI3	PE-CF594	Inhibitory Receptors

OMIP-035

Functional analysis of natural killer cell subsets in macaques

Cytometry PART A, Volume 89, Issue 9, 799–802 (2016)

Kim L. Weisgrau, Moritz Ries, Nicholas Pomplun, David T. Evans, Eva G. Rakasz

Description: This panel was developed to measure the functional capability of natural killer (NK) cell subsets in rhesus macaques (Macaca mulatta). It includes markers to determine the frequency of cytokine secreting and cytotoxic NK cell subpopulations in peripheral blood mononuclear cell (PBMC) samples stimulated in vitro with human 721.221 cells. NK cell subsets were defined by the expression of killer cell immunoglobulin-like receptors (KIRs) Mamu-KIR3DL01 and Mamu-KIR3DL05, and differentiation antigens CD16 and CD56. The panel can be used to assess the functional capability of NK cells in a range of normal and pathologic conditions of captive bred rhesus macaques of Indian origin.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: SORP BD LSR 2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Macaca mulatta		Live/Dead Fix Near IR (775)	viability
CD158	Macaca mulatta	NKVFS1	PE	NK subset
KIR3dl05	Macaca mulatta		APC	NK subset
CD3	Macaca mulatta	SP34-2	PE-CF594	Exclusion
CD8	Macaca mulatta	RPA-T8	Brilliant Violet 711	NK subset
CD16	Macaca mulatta	3G8	Pacific Blue	NK subset
CD20	Macaca mulatta	2H7	PE-CF594	Exclusion
CD45	Macaca mulatta	D058-1283	Brilliant Violet 786	hematopoitic cell lineage
CD56	Macaca mulatta	B159	PerCP-Cy5.5	NK subset
CD107a	Macaca mulatta	H4A3	Brilliant Violet 605	NK cell function
KLRC1/2	Macaca mulatta	Z199	PE-Cy7	NK subset
Granzyme B	Macaca mulatta	GB11	Brilliant Violet 510	NK cell function
IFN-gamma	Macaca mulatta	4S.B3	FITC	NK cell function
TNF alpha	Macaca mulatta	Mab11	Alexa Fluor 700	NK cell function

OMIP-034

Comprehensive immune phenotyping of human peripheral leukocytes by mass cytometry for monitoring immunomodulatory therapies

Cytometry PART A, Volume 91, Issue 1, 34–38 (2017)

Sabine Baumgart, Anette Peddinghaus, Ursula Schulte-Wrede, Henrik E. Mei, Andreas Grützkau

Description: This OMIP-034 (mass cytometry) comprehensively characterizes live human peripheral blood leukocytes from fresh, erythrocyte-depleted whole blood with a single measurement. Different from existing OMIP, it relies on mass cytometry rather than conventional, fluorescence cytometry and thereby combines 26 different markers in a single staining cocktail. The panel has been optimized with respect to marker selection, antibody clones used, pairing of reporter metal, and antibody on the background of isotope mass-dependent machine sensitivity and antigen abundance, avoiding background signals, and signal spill-over. The panel is designed for monitoring patients' leukocytes during immunomodulatory clinical studies in the field of chronic inflammatory, especially autoimmune diseases, but is likely to serve well in different settings, too. This OMIP-034 captures neutrophils, eosinophils, basophils, monocytes, dendritic cells, T and B lymphocytes and NK cells, and their subsets, and contains a selection of cell activation markers (Table 2). It permits leukocyte analyses in their original complexity without influences from density gradient centrifugation or cryopreservation (Table 1). Blank channels were included for the extension of the panel with up to eight markers of interest. The entire protocol takes 2 days, with 60- to 90-min acquisition time to generate data of ∼5 × 105 cell events.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: Fluidigm CyTOF

Panel details

Marker	Species	Clone	Fluorophore	Purpose	Co-expressions groups	Antigen Density

OMIP-033

A comprehensive single step staining protocol for human T- and B-cell subsets

Cytometry PART A, Volume 89, Issue 7, 629-632 (2016)

Tess Brodie, Kristina Rothaeusler, Mireia Sospedra

Description: LIMITED sample availability is a common problem for research with patient mate-rial, and this factor has hampered phenotypic studies. This work addresses the clearneed for a thorough immunophenotyping panel tailored for biological samples withfew cells. This panel is optimized for staining of both cerebrospinal fluid (CSF) lym-phocytes (containing 10–100,000 cells), as well as whole blood. The usually very lowCSF cell numbers make a one-step staining protocol necessary to minimize cell lossin washing steps. CSF cells must be stained immediately due to the CSF’s low proteincontent (1), which renders cells vulnerable. Ideal CSF samples are no older than 1 hand have no fewer than a total of 10,000 cells. This panel identifies human CD4 andCD8 memory subsets as well as T helper subsets, CD41 CD282 costimulation-independent T cells, B cell memory subsets, and plasma cells. Optimal whole bloodsamples should be no older than 5 h after sample acquisition (due to plasma cellloss). In these conditions, samples contain extremely few dead cells, and due to thenecessity of a one-step staining, we did not include a live-dead cell discriminator.This panel can be successfully performed on frozen PBMCs, but authors then recom-mend inclusion of a live/dead marker, and also it needs to be noted that plasma cellsare sensitive to freeze/thawing.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD4	Human	S3.5	PE-Texas Red	T cell lineage
CD294	Human	BM16	PE	Th subset
CD194	Human	L291H4	APC	Th subset
CD3	Human	Hit3a	Alexa Fluor 700	T cell lineage
CD8	Human	SK1	Brilliant Violet 510	T cell lineage
ccr7	Human	G043H7	Brilliant Violet 421	naïve and memory subset
CD45RA	Human	HI100	Brilliant Violet 711	naïve and memory subset
CCR6	Human	G034E3	Brilliant Violet 785	Th subset
CD19	Human	HIB19	PerCP-Cy5.5	B cell
CD138	Human	MI15	FITC	Plasma cell
CD28	Human	CD28.2	PE-Cy7	purative autoreactive cells
IgD	Human	IA6-2	Brilliant Violet 605	B cell
CD27	Human	O323	APC-Cy7	naïve and memory subset

OMIP-032

Two multi-color immunophenotyping panels for assessing the innate and adaptive immune cells in the mouse mammary gland

Cytometry PART A, Volume 89, Issue 6, 527–530 (2016)

Ashleigh Unsworth, Robin Anderson, Nicole Haynes, Kara Britt

Description: A multi-color antibody panel was designed and optimized to identify and characterize 10 leukocyte subpopulations from both the innate and adaptive arms of the immune system. Markers to detect B-cells (CD45.2+ CD19+), T-cells (CD45.2+ TCRβ+), natural killer cells (CD45.2+ TCRβ− CD49b+ NKp46+), and myeloid cells (CD45.2+ CD11b+) were employed. Myeloid cells were further differentiated as dendritic cells (CD45.2+ CD11b+ CD11c+ MHCII+), neutrophils (CD45.2+ CD11b+ Ly6G+), macrophages (CD45.2+ CD11b+ Ly6G− Ly6Clow), and monocytes (CD45.2+ CD11b+ Ly6G− Ly6Chigh). T-cell populations were sub-divided into T-helper cells (CD45.2+ TCRβ+ CD8− CD4+), and cytotoxic T-cells (CD45.2+ TCRβ+ CD4− CD8+). T-cell memory/effector status was determined using CD62L and CD44 to distinguish between effector (CD44+ CD62L−), memory (CD44+ CD62L+) and naïve (CD44− CD62L+) T cells. This panel was established for the analysis of collagenase digested mouse (Balb/C) mammary gland, spleen and tumor samples, as well as RBC-lysed whole blood (Table 1).

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa X20

Panel details

Marker	Species	Clone	Fluorophore	Purpose
MHC II	Mouse	M5/114.15.2	Brilliant Violet 711	DC
CD45.2	Mouse	104	PE	leukocyte
Dead cells	Mouse		Propidium Iodide	Viability
Dead cells	Mouse		Fluoro-Gold	Viability
CD45.2	Mouse	104	APC-Cy7	Leukocyte
CD11c	Mouse	HL3	APC	DC
Ly-6G	Mouse	1A8	PE-Cy7	Neutrophil
CD11b	Mouse	M1/70	PE	Myeloid cell
Ly-6C	Mouse	AL-21	Brilliant Violet 421	Monocyte, macrophage
Mannose Receptor	Mouse	C068C2	FITC	alternatively activated macrophage
CD4	Mouse	Gk1.5	APC-Cy7	Th
TCR beta	Mouse	H57-597	PE-Cy7	T cell
CD49b	Mouse	DX5	FITC	NK
CD8	Mouse	53-6.7	Brilliant Violet 711	cytotoxic T cell
CD44	Mouse	IM7	Brilliant Violet 605	T cell differentiation
CD62L	Mouse	Mel-14	Brilliant Violet 510	T cell differentiation
CD19	Mouse	1D3	APC	B cell
NKp46	Mouse	29A1.4	Brilliant Violet 421	NK

OMIP-031

Immunologic checkpoint expression on murine effector and memory T-cell subsets

Cytometry PART A, Volume 89, Issue 5, 427–429 (2016)

Satoshi Nemoto, Adam W. Mailloux, Jodi Kroeger, James J. Mulé

Description: This panel was designed to assess the expression levels of cell surface inhibitory receptors known as “immune checkpoints” within the context of multiple naïve, activated, memory, and effector phenotypes among T-cells for subsequent adoptive transfer using the CD45.1/CD45.2 congenic system in C57BL/6 mice. It can be easily adapted to other congenic systems, or may be used without any congenic marker. While many panels have been published that analyze T-cell activation, memory phenotypes, or effector differentiation states, few, if any, are comprehensive enough to assess these compartments simultaneously while measuring inhibitory immune checkpoint receptor expression. The ability to do so within a congenic system creates a powerful tool for investigating the evolution of T-cell based immune responses in a broad range of contexts. Here, the panel is used to analyze the T-cell compartment in normal spleen, or T-cells infiltrating subcutaneous murine colon adenocarcinoma, MC38. However, any murine source of T-cells would serve as an appropriate sample source for this panel.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Lag-3	Mouse	C9B7W	Brilliant Violet 711	Checkpoint
PD-1	Mouse	J43	Brilliant Violet 605	Checkpoint
CD127	Mouse	SB/199	Brilliant Ultraviolet 737	Differentiation
CD4	Mouse	Gk1.5	Brilliant Ultraviolet 805	TH
CD44	Mouse	IM7	Alexa Fluor 488	Memory
CD45RA	Mouse	14.8	Brilliant Violet 786	Memory
Dead cells	Mouse		DAPI	Viability
CD3	Mouse	145-2C11	Brilliant Ultraviolet 395	T-cell
CD8	Mouse	53-6.7	Alexa Fluor 700	TC
CD69	Mouse	H1.2F3	PE-CF594	Activation
CD27	Mouse	LG.3A10	Brilliant Violet 510	Memory
CD62L	Mouse	Mel-14	PE-Cy7	Memory
KLRG1	Mouse	2F1/KLRG1	PerCP-Cy5.5	Differentiation
CTLA-4	Mouse	UC10-4B9	PE	Checkpoint
TIM-3	Mouse	B8.2C12	APC	Checkpoint
CD45.2	Mouse	104	APC-Cy7	Congenic

OMIP-030

/omips

Description:

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CCR10	Human	314305	APC	CD4+ T cell subset
CD8	Human	RPA-T8	eFluor 650NC	T cell lineage
Dead cells	Human		Live/Dead Fix Blue	viability
CD38	Human	HB7	eFluor 450	Activation marker
CD4	Human	SK3	Brilliant Violet 510	T cell lineage
CD45RA	Human	HI100	Brilliant Violet 570	naïve/memory
CD3	Human	UCHT1	Qdot 605	T cell lineage
CD25	Human	2A3	Brilliant Violet 711	Treg
CCR6	Human		Brilliant Violet 786	CD4+ T cell subset
CD183	Human	1C6	Alexa Fluor 488	CD4+ T cell subset
CD161	Human	HP-3G10	PerCP-Cy5.5	mNKT, MAIT
CD194	Human	1G1	PE	CD4+ T cell subset
ccr7	Human	150503	PE-CF594	CD4+ T cell subset
CD20	Human	2H7	PE-Cy5.5	B cell exclusion
CD185	Human	RF8B2	Biotin	CD4+ T cell subset
Streptavidin	Human		PE-Cy7	secondary reagent to CD185
Ki-67	Human	B56	Alexa Fluor 700	Proliferation
CD127	Human	RDR5	APC-eFluor 780	Treg

OMIP-030

Characterization of human T cell subsets via surface markers

Cytometry PART A, Volume 87, Issue 12, 1067–1069 (2015)

Gerhard Wingender, Mitchell Kronenberg

Description: The present panel was optimized to quantify the relative frequency of the majority of the major T cell subsets currently described within human peripheral blood mononuclear cells (PBMCs) via the use of surface markers (Table 1). This includes all CD4+ T subsets that received a T helper—nomenclature to date and Tregs. Furthermore, a surrogate staining strategy for the identification of mNKT/MAIT cells without the need of Vα7.2 is proposed. The panel has been validated for fresh and cryopreserved PBMCs. Other tissue types have not been tested.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose	Co-expressions groups	Antigen Density

OMIP-029

Human NK-cell phenotypization

Cytometry PART A, Volume 87, Issue 11, 986-988 (2015)

Yolanda D. Mahnke, Margaret H. Beddall, Mario Roederer

Description: The present panel was optimized to enumerate natural killer (NK) cells within peripheral blood mononuclear cells (PBMC) and to determine their phenotype in terms of NK receptor and differentiation marker expression in healthy individuals. It works well with cryopreserved PBMC and we have observed similar results with fresh specimens. Other tissue types have not been tested (Table 1).

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
NKp46	Human	BAB281	PE-Cy5	NK receptor
CD62L	Human	SK11	Alexa Fluor 680	Differentiation
ccr7	Human	150503	PE-CF594	Differentiation
CD3	Human	SK7	APC-H7	exclusion
CD4	Human	OKT4	Brilliant Violet 605	exclusion
CD16	Human	3G8	Brilliant Violet 421	NK
CD56	Human	HCD56	Brilliant Violet 570	NK
CD158	Human	HP-MA4	FITC	NK receptor
CD158b	Human	DX27	PE	NK receptor
CD314	Human	1D11	PE-Cy7	NK receptor
CD337	Human	p30-15	Alexa Fluor 647	NK receptor
Dead cells	Human		Live/Dead Fix Aqua	Viability/exclusion
CD2	Human	S5.5	PE-Cy5.5	NK

OMIP-028

Activation panel for Rhesus macaque NK cell subsets

Cytometry PART A, Volume 87, Issue 10, 890-893 (2015)

Nicholas Pomplun, Kim L. Weisgrau, David T. Evans, Eva G. Rakasz

Description: This panel was developed to quantify natural killer (NK) cell subsets in Rhesus macaques (Macaca mulatta) during SIVmac239 infection induced pathogenesis. It includes markers to monitor changes in the activation/proliferation phenotype of up to 12 NK cell populations. The performance of the staining was tested on cryopreserved lymph node samples, and on fresh and cryopreserved peripheral blood mononuclear cells (PBMC) isolated from EDTA anti-coagulated blood. The panel can be used to characterize NK cells in a range of normal and pathologic conditions of this species and can be easily adapted to stain samples from various tissues.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Macaca mulatta		Live/Dead Fix Near IR (775)	viability/exclusion
CD158	Macaca mulatta	NKVFS1	PE	NK subset
KIR3dl05	Macaca mulatta		Brilliant Violet 421	NK subset
NKp46	Macaca mulatta	BAB281	PE-Cy5	NK differentiation
CD3	Macaca mulatta	SP34-2	Alexa Fluor 700	exclusion
CD8	Macaca mulatta	SK1	Brilliant Violet 510	NK subset
CD14	Macaca mulatta	M5E2	Alexa Fluor 700	exclusion
CD16	Macaca mulatta	3G8	Brilliant Violet 711	exclusion
CD20	Macaca mulatta	2H7	Alexa Fluor 700	exclusion
CD45	Macaca mulatta	D058-1283	Brilliant Violet 786	hematopoietic cell lineage
CD56	Macaca mulatta	B159	FITC	NK subset
CD69	Macaca mulatta	TP1.55.3	ECD	Activation
KLRC1/2	Macaca mulatta	Z199	PE-Cy7	NK subset
Ki-67	Macaca mulatta	B56	Alexa Fluor 647	Proliferation
HLA-DR	Macaca mulatta	L243	Brilliant Violet 650	Activation
PD-1	Macaca mulatta	EH12.2H7	Brilliant Violet 605	NK differentiation

OMIP-027

Functional analysis of human natural killer cells

Cytometry PART A, Volume 87, Issue 9, 803-805 (2015)

Margaret C. Costanzo, Matthew Creegan, Kerri G. Lal, Micheal A. Eller

Description: The current panel was developed to characterize the function of human natural killer (NK) cells from cryopreserved peripheral blood mononuclear cells (PBMC). The application of this panel is to identify changes in bulk NK cells and NK cell subsets with regard to receptor expression, and function in the setting of acute human immunodeficiency virus (HIV-1) infection. However, this panel may be applied to a wide variety of disease states and normal conditions to characterize human NK cells (Table 1). The performance of this panel was optimized using frozen PBMC from HIV-infected and uninfected individuals. The panel is being used to evaluate NK cell responses in individuals with acute HIV-1 infection as well as normal healthy individuals participating in HIV vaccine clinical trials.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose	Co-expressions groups	Antigen Density

OMIP-026

Phenotypic analysis of B and plasma cells in rhesus macaques

Cytometry PART A, Volume 87, Issue 9, 800-802 (2015)

Berit Neumann, Sieghart Sopper, Christiane Stahl-Hennnig

Description: Our purpose was a broad phenotypic analysis of B and plasma cells regarding differentiation status, activation, proliferation, and chemokine receptor expression in rhesus macaques. We developed two staining panels, which were tested on fresh samples of whole blood or peripheral blood mononuclear cells (PBMCs), bone marrow collected from the iliac crest and femur, lymph nodes, spleen, and tonsils (Figure 1, Table 1). A 10-color-panel was developed to mainly focus on B cells, whereas a 11-color panel concentrated on plasmablasts/plasma cells. Both panels are also applicable for whole blood and bone marrow samples from African green monkeys.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD45	Rhesus macaque		V500	Leukocyte (backbone)
CD10	Rhesus macaque	HI10a	APC-Cy7	Maturation marker (backbone)
CD3	Rhesus macaque	SP34-2	Alexa Fluor 700	Lineage (backbone)
CD20	Rhesus macaque	L27	PE-Cy7	Lineage (backbone)
CD21	Rhesus macaque	B-ly4	FITC	Differentiation (B cell)
CD27	Rhesus macaque	M-T271	APC	Differentiation (B cell)
CD69	Rhesus macaque	TP1.55.3	ECD	Activation (B cell)
CD80	Rhesus macaque	L307.4	PE	Activation (B cell)
ccr7	Rhesus macaque	G043H7	Brilliant Violet 421	Homing (B cell)
IgD	Rhesus macaque	polyclonal	Biotin	Ig (B cell)
CD19	Rhesus macaque	J3-119	PE	Lineage (plasma cell)
CD27	Rhesus macaque	O323	Brilliant Violet 650	Differentiation (plasma cell)
CD38	Rhesus macaque	OKT10	APC	Differentiation (plasma cell)
CD138	Rhesus macaque	DL-101	FITC	Plasma (plasma cell)
CD95	Rhesus macaque	Dx2	Biotin	Activation (plasma cell)
CD184	Rhesus macaque	12G5	PE-CF594	Homing (plasma cell)
Ki-67	Rhesus macaque	B56	PerCP-Cy5.5	Proliferation (plasma cell)
Biotin	Rhesus macaque		Brilliant Violet 570	secondary reagent for CD95 and IgD (plasma cell)

OMIP-025

Evaluation of human T- and NK-cell responses including memory and follicular helper phenotype by intracellular cytokine staining

Cytometry PART A, Volume 87, Issue 4, 289-292 (2015)

Gemma Moncunill, Carlota Dobano, M. Juliana McElrath, Stephen C. Rosa

Description: This panel was developed to assess antigen-specific T cells using peptide pools to various antigens of interest, although other types of antigens such as recombinant proteins or whole pathogens could be considered using different stimulation times. In addition to multiple functional markers, the panel includes differentiation markers and markers to assess follicular helper T cells and NK cells (Table 1). It was optimized using cryopreserved peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV) uninfected and HIV infected adults with known cytomegalovirus (CMV) responses and it underwent assay qualification. The panel is being used to evaluate the responses to HIV and malaria vaccine candidates in adults and children from different geographic areas.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
IL-21	Human	3A3-N2	APC	Function
Dead cells	Human		Live/Dead Fix Aqua	viability
CD4	Human	SK3	Brilliant Ultraviolet 395	T cell lineage
IFN-gamma	Human	B27	V450	Function
CD14	Human	M5E2	Brilliant Violet 510	monocyte (dump)
CD3	Human	UCHT1	Brilliant Violet 570	T cell lineage
CD40L	Human	24-31	Brilliant Violet 605	Function
CD56	Human	HCD56	Brilliant Violet 650	NK, NKT-like cells
CD8	Human	RPA-T8	Brilliant Violet 711	T cell lineage
ccr7	Human	G043H7	Brilliant Violet 785	memory/differentiation
TNF alpha	Human	Mab11	FITC	Function
IL-4	Human	MP4-25D2	PerCP-Cy5.5	Function
IL-2	Human	MQ1-17H12	PE	Function
CD185	Human	MU5UBEE	PE-eFluor 610	Tfh
PD-1	Human		PE-Cy7	Tfh
CD45RA	Human	HI100	APC-H7	memory/differentiation

OMIP-024

Pan-leukocyte immunophenotypic characterization of PBMC subsets in human samples

Cytometry PART A, Volume 85, Issue 12, 995-998 (2014)

Gemma Moncunill, Hannah Han, Carlota Dobano, M. Juliana McElrath, Stephen C. De Rosa

Description: This phenotyping panel was developed to measure the relative frequencies of multiple leukocyte cell subsets in peripheral blood mononuclear cells (PBMC) from African infants and children, including the expression of immune activation and differentiation markers. It was optimized with the objective of obtaining the maximum information concerning the immune status and cell subsets that could influence the immune response to vaccines and infectious diseases using small volumes of pediatric samples. It was developed using cryopreserved PBMC from healthy HIV-uninfected and HIV-infected US adults, but it has also been tested with cryopreserved PBMC from US infants. Although we have not tested the panel on whole blood, it is likely that the panel could be used with whole blood following minimal optimization.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Aqua	viability
CD4	Human	SK3	Brilliant Ultraviolet 395	T cell
CD19	Human	SJ25C1	Brilliant Ultraviolet 737	B cell
CD25	Human	M-A251	Brilliant Violet 421	Treg
HLA-DR	Human		Brilliant Violet 570	Activation
CD56	Human	HCD56	Brilliant Violet 605	NK, NKT-like cell
CD45RA	Human	HI100	Brilliant Violet 650	memory,differentiation
CD14	Human	MfP9	Brilliant Violet 711	monocyte
ccr7	Human	G043H7	Brilliant Violet 785	n/a
CD57	Human	NK-1	FITC	n/a
CD8	Human	SK1	PerCP-Cy5.5	T cell lineage
TCR V delta 2	Human	B6	PE	?d T cell
CD3	Human	UCHT1	ECD	T cell lineage
CD38	Human	HIT2	PE-Cy5	Activation, plasmablasts
TCR gamma/delta	Human	11F2	PE-Cy7	?d T cell
CD127	Human	A019D5	APC	Treg, memory, differentiation
CD159c	Human	134591	Alexa Fluor 700	NK receptor
CD16	Human	3G8	APC-Cy7	NK, monocyte

OMIP-023

10-Color, 13 antibody panel for in-depth phenotyping of human peripheral blood leukocytes

Cytometry PART A, Volume 85, Issue 9, 781-784 (2014)

Jozsef Bocsi, Susanne Meizer, Ingo Dahnert, Attila Tarnok

Description: This panel was developed and optimized to determine the phenotype and activation of 15 different leukocyte subtypes in one run. Leukocytes are identified by expression of CD45 (leu-1) pan leukocyte antigen. Neutrophil (CD16), monocyte (CD14), T- (CD3), B-lymphocyte (CD19), and NK-cell (CD16 and CD56) markers are employed. Special gating strategy is used for subtyping of granulocytes (e.g., eosinophils, neutrophils, basophils). For further T-cell phenotyping, CD4/CD8 markers are used for differentiation of four T-cell subtypes, CD25/CD127 for regulatory T cell identification and CD3/CD16/CD56 for NKT-cells, additionally. Special gating strategies have been developed for B-cell, NK-cell, and monocyte subtyping. For detection and analysis of activation also further activation markers (HLA-DR, CD38, CD25, CD127, and CD69) are analyzed. This panel has been established for analysis of human RBC-lysed EDTA-treated whole blood samples and for cord blood (Table 1). Since the starting material is fresh EDTA-treated blood, dead cells are probably not an issue. Thus to save one channel for specific staining the vitality staining was not used in the panel.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: Beckman Coulter Navios

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD56	Human		PE-Cy7	NK
CD8	Human	B9.11	FITC	CD8 T cell
CD14	Human	RMO52	FITC	Monocyte
CD19	Human	J3-119	FITC	B-cell differentiation
CD69	Human	TP1.55.3	PE	Activation
CD25	Human	B1.49.9	ECD	Treg
CD38	Human	LS198.4.3	PE-Cy5.5	B-cell differentiation
CD16	Human	3G8	PE-Cy7	Monocyte, NK
HLA-DR	Human	Immu-357	APC	Monocyte, NK gating
CD127	Human	R34.34	APC-Alexa 700	Treg gating
CD4	Human	SK3	APC-H7	CD4 T cell
CD45	Human	J.33	Pacific Blue	Leukocyte
CD3	Human	SP34-2	V500	T cell

OMIP-022

Comprehensive assessment of antigen-specific human T-cell functionality and memory

Cytometry PART A, Volume 85, Issue 7, 576-579 (2014)

Andrew J. Graves, Marcelino G. Padilla, David A. Hokey

Description: This flow cytometry antibody panel was developed and optimized for the characterization of CD4+ and CD8+ T-cell memory and functional responses in adult and infant cryopreserved peripheral blood mononuclear cells (PBMC) stimulated with peptide pools to various antigens of interest. The panel has been used to evaluate Mycobacterium tuberculosis (TB) antigen-specific responses in clinical trial specimens, and is currently undergoing assay qualification.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Aqua	viability
CD14	Human	M5E2	V500	exclusion
CD19	Human	HIB19	V500	exclusion
CD3	Human	UCHT1	ECD	T cell
CD4	Human	RPA-T4	APC-eFluor 780	T cell subset
CD8	Human	Hit8a	Alexa Fluor 700	T cell subset
ccr7	Human	G043H7	Brilliant Violet 605	Memory
CD45RO	Human	UCHL1	Brilliant Violet 785	Memory
IFN-gamma	Human	B27	V450	Th1
IL-2	Human	MQ1-17H12	PE	Th
TNF alpha	Human	Mab11	PE-Cy7	Th
IL-17A	Human	BL168	PerCP-Cy5.5	Th17
IL-22	Human	IL22JOP	APC	Th22
CD107a	Human	H4A3	Alexa Fluor 488	Degranulation
CD40L	Human	TRAP1	PE-Cy5	Activation/B-Cell help

OMIP-021

Simultaneous quantification of human conventional and innate-like T-cell subsets

Cytometry PART A, Volume 85, Issue 7, 573-575 (2014)

Nicholas A. Gherardin, David S. Ritchie, Dale I. Godfrey, Paul J. Neeson

Description: This panel was developed in order to simultaneously quantify both conventional peptide-MHC-restricted, and innate-like T-cell compartments in human peripheral blood samples. The panel can assess the dynamics of naïve through to terminally differentiated effector memory T-cell subsets, as well as enumerating natural killer T (NKT) cells, mucosal-associated invariant T (MAIT) cells, γδ T-cells, and subsets thereof. The panel is suitable for use on both, freshly isolated or cryopreserved peripheral blood mononuclear cells (PBMC). Staining may be performed in a 96-well plate to increase throughput.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
ccr7	Human	3D12	PE-Cy7	memory T cell subset
CD45RA	Human	HI100	PerCP-Cy5.5	memory T cell subset
Dead cells	Human		Live/Dead Fix Aqua	Viability (dump)
CD3	Human	OKT3	Brilliant Violet 785	Lineage
CD4	Human	RPA-T4	APC-Cy7	Phenotyping
CD8a	Human	RPA-T8	Brilliant Violet 650	Phenotyping
CD8b	Human	2ST8.5H7	APC	Phenotyping
CD27	Human	O323	Brilliant Violet 711	memory T cell subset
CD28	Human	28.2	PE-Cy5	memory T cell subset
CD45RO	Human	UCHL1	Alexa Fluor 700	memory T cell subset
CD161	Human	HP-3G10	Brilliant Violet 605	MAIT
TCR gamma/delta	Human	11F2	FITC	?d T cell
TCR V alpha 7.2	Human	3C10	PE	MAIT
Streptavidin	Human		Brilliant Violet 421	NKT

OMIP-020

Phenotypic characterization of human γδ T-cells by multicolor flow cytometry

Cytometry PART A, Volume 85, Issue 6, 522-524 (2013)

Kilian Wistuba-Hamprecht, Graham Pawelec, Evelyna Derhovanessian

Description: This panel was composed and optimized to investigate the differentiation stages of human γδ T-cells in cryopreserved peripheral blood mononuclear cells (PBMC) from healthy individuals (Tables 1 and 2). As the majority of pan-γδ T-cell antibodies available commercially proved to be inappropriate for detecting all γδ T-cell populations in combination with other markers, this panel provides an essential tool for the analysis of different subsets of human γδ T-cells by flow cytometry. The panel works very well with cryopreserved PBMC. Other tissues have not been tested.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		EMA	viability
CD3	Human	UCHT1	Alexa Fluor 700	Lineage
CD4	Human	OKT4	PE-Cy7	CD4 T cell
CD8	Human	SK1	APC-H7	CD8 T cell
CD16	Human	3G8	Brilliant Violet 711	?dT activation
CD27	Human		APC	differentiation
CD28	Human	CD28.2	PE	differentiation
CD45RA	Human		Pacific Blue	differentiation
TCR gamma/delta	Human	11F2	Purified	?dT cell
IgG	Human	N/A	Pacific Orange	secondary reagent to ?d-TCR (clone qqF2)
TCR V delta 1	Human	TS8.2	FITC	?dT cell subset
TCR V delta 2	Human	B6	PerCP	?dT cell subset

OMIP-019

Quantification of human γδT-cells, iNKT-cells, and hematopoietic precursors

Cytometry PART A, Volume 83A, Issue 8, 676-678 (2013)

Yolanda D. Mahnke, Margaret H. Beddall, Mario Roederer

Description: The present panel was optimized to quantify the relative frequencies of γδT-cells, invariant natural killer T-cells (iNKT-cells), and hematopoietic precursors in peripheral blood mononuclear cells (PBMC) from healthy individuals (Table 1). It works well with cryopreserved PBMC and we have observed similar results with fresh specimens. Other tissue types have not been tested.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
TCR V delta 1	Human	TS8.2	FITC	?dT cell
ccr7	Human	150503	Alexa Fluor 680	phenotyping
CD1d	Human		PE	iNKT
CD3	Human	OKT3	Brilliant Violet 785	lineage
CD27	Human		Qdot 655	phenotyping
TCR V delta 2	Human	B6	Alexa Fluor 594	?dT cell
Dead cells	Human		Live/Dead Fix Aqua	viability
CD28	Human	CD28.2	PE-Cy5	Surface
CCR5	Human	2D7/CCR5	APC-Cy7	phenotyping
CD4	Human	OKT4	Brilliant Violet 605	phenotyping
CD34	Human	HI100	Brilliant Violet 421	Hematopoietic stem cell
TCR V gamma 9	Human	B3	APC	?dT cell
CD45RA	Human	MEM-56	PE-Cy5.5	phenotyping
CD8	Human	RPA-T8	Qdot 585	phenotyping

OMIP-018

Chemokine receptor expression on human T helper cells

Cytometry PART A, Volume 83A, Issue 6, 530-532 (2013)

Tess Brodie, Elena Brenna, Federica Sallusto

Description: This panel was developed for the enumeration of chemokine receptor expression on CD4 T cells from the naïve (TN), stem cell memory (TSCM), central memory (TCM), and effector memory (TEM) cell subsets (Table 1). Eight chemokine receptors were chosen based upon previously published observations implicating their preferential expression on T helper type 1 (Th1), Th2, Th17, or Th22 cells, and their role in controlling lymphocyte migration to secondary lymphoid tissues, B cell follicles or non-lymphoid tissues, both in the steady state and during immune responses to pathogens, autoantigens, or allergens (1). This panel has been optimized for use with fresh peripheral blood mononuclear cells (PBMCs) with the observation that 24 h after blood draw, the expression of some chemokine receptors is reduced. Optimal staining requires PBMCs to be processed and acquired within 5 h of blood draw and may be performed in a 96-well plate format for high throughput experiments.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD185	Human	51505	Qdot 605	polarization
CD95	Human	Dx2	PerCP-eFluor 710	maturity
Biotin	Human		Qdot 800	secondary reagent to CCR6
ccr7	Human	G043H7	Brilliant Violet 421	maturity
CD194	Human	1G1	PE-Cy7	polarization
CD183	Human	1C6/CXCR3	PE-Cy5	polarization
CCR5	Human	2D7/CCR5	APC-Cy7	polarization
CD193	Human	61828	APC	polarization
CD294	Human	BM16	FITC	polarization
CD3	Human		PE-Cy5.5	T cell
CD4	Human	S3.5	PE-Texas Red	T cell
CD45RA	Human	MEM-56	Qdot 655	maturity
CCR6	Human	11A9	Biotin	polarization
CCR10	Human	314305	PE	polarization
CD14	Human	M5E2	V500	exclusion
CD16	Human	3G8	V500	exclusion
CD19	Human	HIB19	V500	exclusion
Dead cells	Human		Live/Dead Fix Aqua	viability/exclusion

OMIP-017

Human CD4+ helper T-cell subsets including follicular helper cells

Cytometry PART A, Volume 83A, Issue 5, 439-440 (2013)

Yolanda D. Mahnke, Margaret H. Beddall, Mario Roederer

Description: This panel was optimized to measure the relative frequencies of CD4+ T-helper cell subsets and follicular helper T-cells in peripheral blood mononuclear cells (PBMC) from healthy individuals (Table 1). It works well with cryopreserved PBMC and we have observed similar result with fresh specimens. Other tissue types have not been tested.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
ccr7	Human	150503	Alexa Fluor 680	differentiation
CD194	Human		PE-Cy7	Th subset (chemokine leve)
CD3	Human	UCHT1	Alexa Fluor 594	lineage
CD4	Human	RPA-T4	Qdot 800	lineage
CD45RA	Human	5H9	Qdot 655	differentiation
Dead cells	Human		Live/Dead Fix Aqua	viability
CCR6	Human	G034E3	Brilliant Violet 605	Th subset (chemokine leve)
CCR10	Human	6588-5	PE	Th subset (chemokine leve)
CD183	Human	1C6/CXCR3	PE-Cy5	Th subset (chemokine leve)
CD185	Human	RF8B2	Alexa Fluor 647	Tfh
PD-1	Human	EH12.2H7	Brilliant Violet 421	exploratory
CD8	Human	RPA-T8	Qdot 585	lineage
CD161	Human	DX12	FITC	exploratory

OMIP-016

Characterization of antigen-responsive macaque and human T-cells

Cytometry PART A, Volume 83A, Issue 2, 182-184 (2013)

Sabrina Guenounou, Nathalie Bosquet, Claudia J. Dembek, Roger Le Grand, Antonio Cosma

Description: The present panel was optimized to assess the quality and phenotype of antigen-specific CD4 and CD8 T cells in both cynomolgus macaques and humans. The use of an identical protocol for specimens collected in the two species allows for an immediate translation of research from the macaque model to humans. Our protocol works well with cryopreserved and freshly collected PBMC. Following the fixing and permeabilization procedure, we introduced a freezing step that breaks the experimental procedure and allows the shipment of freshly stimulated and permeabilized samples to facilities equipped with instruments able to measure ten distinct fluorescences. Our procedure is thus adapted to multicenter studies where stimulation is performed on fresh PBMC and flow cytometry acquisition is done in a centralized facility.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD45RA	Cynomolgus macaque	L48	PE-Cy7	Differentiation
Dead cells	Cynomolgus macaque		Live/Dead Fix Blue	viability
CD3	Cynomolgus macaque	SP34-2	APC-Cy7	T cell subset
CD4	Cynomolgus macaque	L200	PerCP-Cy5.5	T cell subset
CD8	Cynomolgus macaque	RPA-T8	V500	T cell subset
CD40L	Cynomolgus macaque	TRAP1	FITC	Cytokines/Chemokine/Activation marker
MIP-1 beta	Cynomolgus macaque	D21-1351	PE	Cytokines/Chemokine/Activation marker
IFN-gamma	Cynomolgus macaque	B27	V450	Cytokines/Chemokine/Activation marker
TNF alpha	Cynomolgus macaque	Mab11	Alexa Fluor 700	Cytokines/Chemokine/Activation marker
IL-2	Cynomolgus macaque	MQ1-17H12	APC	Cytokines/Chemokine/Activation marker

OMIP-015

Human regulatory and activated T-cells without intracellular staining

Cytometry PART A, Volume 83A, Issue 2, 179-181 (2013)

Yolanda D. Mahnke, Margaret H. Beddall, Mario Roederer

Description: The present panel was optimized to investigate the frequency and phenotype of regulatory T-cells (Treg), as well as the activation status of CD4+ and CD8+ T-cells in peripheral blood mononuclear cells (PBMC) from healthy individuals, without the use of intracellular staining (i.e., excluding the use of the canonical Treg marker, FoxP3). The panel has been developed using cryopreserved PBMC and we have observed similar results with fresh specimens. Other tissue types have not been tested.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD4	Human	OKT4	Qdot 605	lineage
CD8	Human	RPA-T8	Qdot 585	lineage
CD45RA	Human	5H9	Qdot 705	activation/differentiation
HLA-DR	Human	G46-6	Alexa Fluor 680	activation/differentiation
Dead cells	Human		Live/Dead Fix Aqua	viability
CD25	Human	M-A251	PE-Cy5	Treg
CD38	Human	HIT2	PE-CF594	activation/differentiation
CD39	Human		PE-Cy7	Treg function
CD45RO	Human	UCHL1	FITC	activation/differentiation
CD73	Human	AD2	PE	Treg function
CD127	Human	eBioRDR5	APC-eFluor 780	Treg
PD-1	Human	EH12.2H7	Brilliant Violet 421	activation/differentiation

OMIP-014

Validated multifunctional characterization of antigen-specific human T cells by intracellular cytokine staining

Cytometry PART A, Volume 81A, Issue 5, 362-363 (2012)

Stephen C. De Rosa, Donald K. Carter, M. Juliana McElrath

Description: This panel was developed, optimized, and validated for assessment of CD4+ and CD8+ T-cell responses to various peptide pools for antigens of interest in cryopreserved peripheral blood mononuclear cells (PBMC) from adult humans (Table 1). The panel has been used to evaluate HIV- and TB-specific responses to candidate vaccines for these pathogens, although the panel can be used with peptide pools for any proteins. The panel has not been tested with freshly-isolated PBMC or with whole blood.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Aqua	viability
CD14	Human	Tük4	Qdot 655	exclusion
IFN-gamma	Human	B27	V450	function
IL-2	Human	MQ1-17H12	PE	function
TNF alpha	Human	Mab11	FITC	function
IL-4	Human	MP4-25D2	APC	function
MIP-1 beta	Human	D21-1351	Alexa Fluor 700	function
CD40L	Human	TRAP1	PE-Cy5	function
CD107a	Human	H4A3	PE-Cy7	function
CD3	Human	UCHT1	PE-Texas Red	lineage
CD4	Human	13B8.2	APC-Alexa 750	lineage
CD8	Human	SK1	PerCP-Cy5.5	lineage

OMIP-013

Differentiation of human T-cells

Cytometry PART A, Volume 81A, Issue 11, 935-936 (2012)

Yolanda D. Mahnke, Margaret H. Beddall, Mario Roederer

Description: The present panel was optimized to investigate the differentiation status of CD4+ and CD8+ T-cells in peripheral blood mononuclear cells (PBMC) fromhealthy individuals. It works well with cryopreserved PBMC and we have observed similar results with fresh specimens. Other tissue types have not been tested.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
ccr7	Human	150503	Alexa Fluor 680	memory/differentiation
CD244	Human	C1.7	PE-Cy5.5	memory/differentiation (exploratory)
CD8	Human	RPA-T8	Qdot 585	T-cell subset
CD4	Human	OKT4	Qdot 605	T-cell subset
CD57	Human	NK-1	Qdot 705	memory/differentiation for TSCM
Dead cells	Human		Live/Dead Fix Aqua	viability
CD127	Human	A019D5	Brilliant Violet 421	memory/differentiation for TSCM
CD95	Human	Dx2	PE	memory/differentiation for RTE and TSCM
CD31	Human	WM59	PE-Cy7	memory/differentiation for RTE and TSCM
CD27	Human	O323	FITC	memory/differentiation
CD28	Human	CD28.2	PE-Cy5	memory/differentiation for TSCM
CD45RA	Human	HI100	APC	memory/differentiation
CD3	Human	SK7	APC-H7	T-cell subset

OMIP-012

Phenotypic and numeric determination of human leukocyte reconstitution in humanized mice

Cytometry PART A, Volume 81A, Issue 8, 646-648 (2012)

Brian R. Long, Cheryl A. Stoddart

Description: This panel was developed to determine both the frequency and absolute number of human leukocytes and leukocyte subsets present in the peripheral blood of humanized mice. The panel also provides information concerning the activation state of peripheral leukocytes by cell surface staining for HLA-DR and CD38, relevant to studies of HIV disease pathogenesis (1–3). This panel has been used with EDTA anticoagulated whole blood in conjunction with bead-based enumeration for quantitative assessment of human cell chimerism. This panel also works well for staining of peripheral blood mononuclear cells (PBMCs) prepared by density gradient centrifugation and for dispersed splenocytes. The multicolor panel described here has been used in studies of humanized mouse reconstitution (4) and longitudinal studies of HIV pathogenesis in NSG-BLT mice (5).

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD19	Human	HIB19	APC-Cy7	human B cell
CD45	Human	HI30	Alexa Fluor 700	human leukocyte
CD45	Mouse	30-F11	APC	excusion of mouse leukocyte
CD56	Human	HCD56	PE-Cy7	human NK
CD3	Human	UCHT1	ECD	human T cell
CD38	Human	HIT2	PE	activation
HLA-DR	Human	L243	FITC	activation
CD8	Human	3B5	Qdot 605	human CD8
CD4	Human	RPA-T4	Pacific Blue	human CD4
Isotype Ctrl	Mouse	MOPC-21	PE	set gate for CD38
CD94	Human	DX22	FITC	NK
CD16	Human	3G8	PerCP	NK
CD314	Human	1D11	PE	NK
CD159c	Human	134591	APC	NK

OMIP-011

Characterization of circulating endothelial cells (CECs) in peripheral blood

Cytometry PART A, Volume 81A, Issue 7, 549-551 (2012)

Raskit Lachmann, Paola Lanuti, Sebastiano Miscia

Description: This panel was optimized for the evaluation of circulating endothelial cells (CECs) in peripheral blood (see Table 1). The combination of three different endothelial cell markers enables a reasonable analysis of CECs. The panel, so far tested on fresh human peripheral and cord blood, can be used to enumerate CECs in a dual platform method.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD FACSCanto 2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD34	Human	581	ECD	endothelial cell marker
DNA	Human		Syto 16	DNA marker
Dead cells	Human		Nile Red	Viability
CD45	Human	HI30	Alexa Fluor 700	Exclusion of leukocyte
CD31	Human	M89D3	Alexa Fluor 647	endothelial cell marker
CD146	Human	P1H12	PE	endothelial cell marker
CD117	Human	104D2	PE-Cy7	progenitor marker
CD106	Human	51-10C9	PE-Cy5	activation

OMIP-010

A new 10-color monoclonal antibody panel for polychromatic immunophenotyping of small hematopoietic cell samples

Cytometry PART A, Volume 81A, Issue 6, 453-455 (2012)

Frank W. M. B. Preijers, Erik Huys, Bijan Moshaver

Description: The 10-color panel consisting of 15 monoclonal antibodies (mAbs) is developed to detect leukemia and lymphoma cells in small cell samples [hypoplastic bone marrow (BM), fine needle aspirates, or cerebral spinal fluids (CSFs)]. MAbs conjugates were selected to identify populations of distinct cell lineages and to determine stages of differentiation based on specific antigen expression patterns. As such, conjugates containing the same fluorochrome could be combined. This panel is tested on peripheral blood (PB), BM, and CSF and provides a strong improvement of diagnostic potential.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: Beckman Coulter Navios

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD19	Human	J3-119	APC-Alexa 750	B
CD33	Human	D3HL60.251	APC	T cell
CD34	Human	581	FITC	lymphoid and meyloid progenitors
CD56	Human		PE-Cy7	NK, NKT
CD4	Human		PE-Cy5.5	T cell
Ig Lambda Light Chain	Human		FITC	secondary reagent to CD34
CD7	Human	8H8.1	PE	T cell
Ig Lambda Light Chain	Human		PE	secondary reagent to CD7
CD10	Human	ALB1	ECD	B cell precursors
CD117	Human		PE-Cy7	lymphoid and meyloid progenitors
CD15	Human	80H5	Pacific Blue	B
CD20	Human	B9E9	Pacific Blue	B
CD45	Human	J33	Krome Orange	lymphocyte
CD3	Human	UCHT1	APC	T cell
CD8	Human	B9.11	APC-Alexa 700	T cell

OMIP-009

Characterization of antigen-specific human T-cells

Cytometry PART A, Volume 81A, Issue 5, 363-363 (2012)

Laurie Lamoreaux, Richard A. Koup, Mario Roederer

Description: The panels described in this article are designed to characterize the immunological response of human T-cells to vaccination by measuring the frequency, phenotype, and function of CD4 and CD8 T-cells. Subsequent qualification of the panel allows for comparison of intra- and inter-laboratory outcomes between different vaccine trials such that those vaccine formulations that reveal a possible correlate of protection against infection may be moved forward in the regulatory process. Although the panel was developed for batch analysis of cryopreserved PBMC samples, the assay may also be performed with fresh cells (Table 1).

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
ccr7	Human	150503	Alexa Fluor 680	memory/differentiation
Dead cells	Human	N/A	Live/Dead Fix Violet	viability
CD3	Human	SP34-2	APC-Cy7	lineage
CD45RA	Human	L48	PE-Cy7	memory/differentiation
CD28	Human		PE-Cy7	memory/differentiation
CD4	Human	T4	ECD	lineage
CD8	Human	RPA-T8	Pacific Blue	lineage
IFN-gamma	Human	B27	APC	function
IL-2	Human	MQ1-17H12	PE	function
TNF alpha	Human	Mab11	FITC	function

OMIP-008

Measurement of Th1 and Th2 cytokine polyfunctionality of human T cells

Cytometry PART A, Volume 81A, Issue 6, 450-452 (2012)

Cindy L. Zuleger, Mark R. Albertini

Description: This panel was optimized to assess CD4+ and CD8+ T cell responses to various tumor antigens from melanoma patients. The panel was tested on single-cell derived T cell isolates (SCD-T) and T cell lines derived from peripheral blood mononuclear cells (PBMC) from melanoma patients, T cell lines from the tumor environment of melanoma patients, and fresh and cryopreserved PBMC (healthy donors). Staining can be performed in 96-well plates for high-throughput.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD19	Human	HIB19	eFluor 450	exclusion
CD3	Human	UCHT1	Alexa Fluor 700	lineage
Dead cells	Human		Live/Dead Fix Violet	viability(dump)
CD8	Human	3B5	Qdot 605	lineage
CD4	Human	RPA-T4	APC-Cy7	lineage
CD14	Human	M5E2	Pacific Blue	exclusion
IL-2	Human	MQ1-17H12	PerCP-Cy5.5	function/cytokine secretion
IL-4	Human	8D4-8	Alexa Fluor 488	function/cytokine secretion
IL-10	Human	JES3-9D7	PE	function/cytokine secretion
IFN-gamma	Human	B27	APC	function/cytokine secretion
TNF alpha	Human	Mab11	PE-Cy7	function/cytokine secretion

OMIP-007

Phenotypic analysis of human natural killer cells

Cytometry PART A, Volume 81A, Issue 6, 447-449 (2012)

Micheal A. Eller, Jeffrey R. Currier

Description: This panel was developed to characterize the phenotype of human natural killer (NK) cells from cryopreserved peripheral blood mononuclear cells (PBMC) isolated from ACD or EDTA anticoagulated whole blood or apheresis units (Table 1). The application of this panel was to identify changes in NK cell subsets with regard to receptor expression, maturation, homing potential, and activation in the setting of primary HIV-1 natural infection. However, this panel may be applied to a wide variety of disease and normal conditions to characterize NK cells in humans. The performance of this panel was tested on fresh and frozen PBMC as well as using a whole blood lyse no wash procedure.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
CD16	Human	3G8	Pacific Blue	NK subset
Integrin alpha 4 beta 7	Human	ACT-1	Qdot 655	Homing
CD62L	Human	DREG56	Qdot 605	Homing
CD57	Human	hCD57	APC	Differentiation
Dead cells	Human		Live/Dead Fix Aqua	Viability
CD3	Human	S4.1	PE-Texas Red	Exclusion
CD4	Human	SFCI12T4D11	ECD	Exclusion
CD14	Human	Tuk4	PE-Cy5	Exclusion
CD19	Human	SJ25-C1	PE-Cy5	Exclusion
CD56	Human	NCAM16.2	PE-Cy7	NK subset
CD8	Human	SK1	APC-H7	NK subset
CD158	Human	HP-MA4	PerCP-Cy5.5	KIR receptor
CD158b1/b2,j	Human	DX27	PE	KIR receptor
CD158e	Human	DX9	Alexa Fluor 700	KIR receptor
HLA-DR	Human	G46-6	FITC	Activation

OMIP-006

Phenotypic subset analysis of human T regulatory cells via polychromatic flow cytometry

Cytometry PART A, Volume 81A, Issue 4, 281-283 (2012)

David M. Murdoch, Janet S. Staats, Kent J. Weinhold

Description: This panel was optimized for the enumeration and phenotypic characterization of T regulatory cells (Tregs) within the CD4+ T-cell pool using human peripheral blood mononuclear cells (PBMC) using intranuclear and intracellular staining methods. The panel was optimized for HIV+ clinical trial specimens through the use of HIV-infected and normal donor PBMC. Because the panel is to be used in the context of testing cryopreserved PBMC obtained from multiple sites participating in clinical trials, it was essential to develop an assay that performed well using cryopreserved PBMC. Other tissue types have not been tested. © 2012 International Society for Advancement of Cytometry.

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Violet	viability
CD45RO	Human	UCHL1	FITC	maturation/differentiation
CD4	Human	SK3	PerCP-Cy5.5	T cell subset
FoxP3	Human	PCH101	PE	Treg identification
CD25	Human	B1.49.9	ECD	T cell activation, Treg identification
CD49d	Human	9F10	PE-Cy5	Treg subset
CD39	Human		PE-Cy7	Treg subset
Helios	Human	22F6	Alexa Fluor 647	Treg subset
CD3	Human	UCHT1	V500	T cell subset

OMIP-005

Quality and phenotype of antigen-responsive rhesus macaque T cells

Cytometry PART A, Volume 81A, Issue 5, 360-361 (2012)

Kathryn E. Foulds, Mitzi Donaldson, Mario Roederer

Description: The primary eight-color panel was designed to measure IFNγ, IL-2, and TNF production from viable CD4 and CD8 T cells from rhesus macaques in preclinical vaccine studies. An 11-color variant also allows for the assessment of memory subsets based on surface expression of CD28, CD45RA, and CCR7. The panel was optimized not only for use on cryopreserved peripheral blood mononuclear cell (PBMC) samples but also works well on fresh PBMC samples, cryopreserved tissue samples, and fresh tissue samples that have been treated with RBC lysis buffer (Table 1). The eight-color panel and associated staining procedure were tested in a formal qualification study and shown to be highly reproducible with low interaliquot, interday, and interanalyst variability according to the qualification criteria (manuscript in preparation).

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Machine: BD LSR2

Panel details

Marker	Species	Clone	Fluorophore	Purpose
ccr7	Rhesus macaque	150503	Alexa Fluor 680	memory
Dead cells	Rhesus macaque		Live/Dead Fix Aqua	viability
CD4	Rhesus macaque	S3.5	Qdot 605	T cell
CD45RA	Rhesus macaque	L48	PE-Cy7	memory
CD28	Rhesus macaque	28.2	PE-Cy5	memory
CD8	Rhesus macaque	RPA-T8	Pacific Blue	T cell
CD3	Rhesus macaque	SP34-2	APC-Cy7	T cell
CD69	Rhesus macaque	TP1.55.3	ECD	background reduction
IFN-gamma	Rhesus macaque	B27	APC	cytokines
IL-2	Rhesus macaque	MQ1-17H12	PE	cytokines
TNF alpha	Rhesus macaque	Mab11	FITC	cytokines

OMIP-004

In-depth characterization of human T regulatory cells

Cytometry PART A, Volume 81A, Issue 1, 15-16 (2012)

Angelique Biancotto, Pradeep K. Dagur, J. Chris Fuchs, Marc Langweiler, J. Philip McCoy Jr

Description: The present panel was constructed for the in-depth characterization of human T regulatory cells in both health and disease. The panel works well in both fresh and cryopreserved PBMCs (Table 1). The panel has also been tested on ACK-lysed peripheral blood. No other types of tissues have been tested.

Species: Human

Tissue: Peripheral blood

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Keyword: Cryopreserved pbmc

Machine: BD LSR Fortessa

Panel details

Marker	Species	Clone	Fluorophore	Purpose
Dead cells	Human		Live/Dead Fix Aqua	Viability
CD45	Human		Qdot 800	leukocyte
CD3	Human	SK7	APC-Cy7	T cell
CD4	Human	RPA-T4	V450	T helper subset
CD8	Human	3B5	Qdot 605	T suppressor subset
CD25	Human	M-A251	PE-Cy7	Treg characterization
CD27	Human	CLB-27/1	Qdot 655	Treg characterization
CD38	Human	HIT2	PerCP-Cy5.5	Treg characterization
CD39	Human	A1	Alexa Fluor 488	Treg supression
CD45RA	Human		PE-Texas Red	naïve T cell
CD103	Human	LF61	PE-Cy5	naïve T cell
CD127	Human	hIL-7R-M21	Alexa Fluor 647	Treg characterization
ccr7	Human	150503	Alexa Fluor 700	Treg characterization
HLA-DR	Human		PE-Cy5.5	Treg characterization
FoxP3	Human	PCH101	PE	Treg marker

OMIP-003

Phenotypic analysis of human memory B cells

Cytometry PART A, Volume 79A, Issue 11, 894-896 (2011)

Chungwen Wei, John Jung, Inaki Sanz

Description: This panel was developed to characterize the phenotypic diversity of human memory B cells, with an emphasis on discriminating cell subsets within both the conventional memory population (CD27+) and the more recently described isotype switched (IgD−) population lacking expression of CD27 (1). It has been tested on fresh and cryopreserved peripheral blood mononuclear cells (PBMC), as well as bone marrow aspirates and tonsillar cells (Table 1). The multicolor panel described herein has been used extensively to analyze large numbers of PBMC samples obtained from healthy controls in steady state and in response to infection (HIV, influenza, respiratory syncytical virus) and vaccination (influenza, tetanus) as well as in hundreds of patients with autoimmune diseases (systemic lupus erythematosus (SLE), rheumatoid arthritis, Sjogren's syndrome, psoriatic arthritis and Type 1 diabetes) and conditions characterized by allogeneic immune responses (renal transplantion and chronic graft versus host disease). This panel is also being applied in a longitudinal study in which 150 SLE patients are to be followed quarterly for a period of 2 years.

Species: Human

Tissue: Bone marrow, Peripheral blood, Tonsil

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Keyword: Cryopreserved pbmc, Hiv, Influenza, Psoriatic arthritis, Respiratory syncytical virus (rsv), Rheumatoid arthritis (ra), Sjogren's syndrome, Type 1 diabetes, Vaccination

Machine: Accuri C6

Panel details

Marker	Species	Clone	Fluorophore	Purpose
IgD	Human	IA6-2	FITC	differentiation
CD183	Human	1C6/CXCR3	PE	homing
CD24	Human	SN3	PE-Alexa 610	differentiation, epithelial adhesion
CD21	Human	B-ly4	PE-Cy5	activation, BCR co-receptor for antigen bound C3d
CD38	Human	HIT2	PerCP-Cy5.5	differentiation
CD45R (B220)	Human		PE-Cy7	differentiation
CD3	Human	SP34-2	Pacific Blue	exclusion
Dead cells	Human		Live/Dead Fix Aqua	exclusion
CD27	Human	CLB-27/1	Qdot 605	differentiation, receptor for CD70 on activated T cell
CD19	Human	SJ25C1	APC-Cy7	lineage
CD95	Human	Dx2	APC	activation, pro-apoptotic in the presence of activated CD95L T cells
Biotin	Human	Streptavidin	Alexa Fluor 680	n/a

OMIP-002

Phenotypic analysis of specific human CD8+ T-cells using peptide-MHC class I multimers for any of four epitopes

Cytometry PART A, Volume 77A, Issue 9, 821-822 (2010)

Pratip K Chattopadhyay, Mario Roederer, David A. Price

Description: This panel was developed to determine the phenotype of human antigen (Ag)-specific CD8+ T-cells. Ag-specificities are identified by four peptide-major histocompatibility complex (MHC) class I (pMHCI) multimers (e.g., against Epstein-Barr virus (EBV) and cytomegalovirus (CMV) epitopes). Six markers of T-cell phenotype are used. This panel has been tested on fresh and cryopreserved peripheral blood mononuclear cells (PBMC), as well as bone marrow samples; staining may be performed in 96-well plates to increase throughput.

Species: Human

Tissue: Bone marrow, Peripheral blood

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Keyword: Antigen-specific t cell response, Cryopreserved pbmc, Cytomegalovirus (cmv), Epstein-barr virus (ebv), Tetramer

Machine: LSR II

Panel details

Marker	Species	Clone	Fluorophore	Purpose	Co-expressions groups	Antigen Density
CD14	Human	M5E2	Pacific Blue	exclusion		High
CD127	Human	R34.34	PE-Cy5.5	maturity	T memory	Med
Biotin	Human		APC	secondary reagent to PD-1
CD45RO	Human	UCHL1	APC-Alexa 700	maturity	T memory	High
CD19	Human	HIB19	Pacific Blue	exclusion		Low
CD4	Human		Qdot 705	T cell subset	T memory	High
ccr7	Human	150503	PE-Alexa 750	maturity
Dead cells	Human		Live/Dead Fix Violet	viability
CD27	Human	1A4	PE-Cy5	maturity	T memory	Med
PD-1	Human	MIH4	Biotin	maturity	T memory	Low
CD3	Human	SK7	APC-Cy7	T cell subset	T memory	High
CD57	Human	NK-1	FITC	maturity	T memory	High

OMIP-001

Quality and phenotype of Ag-responsive human T-cells

Cytometry PART A, Volumn 77A, Issue 9, 819-820 (2010)

Yolanda D. Mahnke, Mario Roederer

Description: The present panel was optimized for the evaluation of CD4+ and CD8+ T-cell responses to various HIV-1–derived peptide pools in peripheral blood mononuclear cells (PBMC) from HIV-1+ individuals with differences in clinical progression. It works well with cryopreserved PBMC, and we have observed similar results with fresh specimens. Other tissue types have not been tested.

Species: Human, Rat

Tissue: Peripheral blood

Cell Subset: T cell, T memory, Treg, Erythroid cells, Erythrocyte, Granulocyte, Basophil, Lymphoid cells, T helper, Megakaryocyte, B cell, B memory

Keyword: Cryopreserved pbmc, Hiv

Machine: LSR II

Panel details

Marker	Species	Clone	Fluorophore	Purpose	Co-expressions groups	Antigen Density
CD14	Human	M5E2	Pacific Blue	exclusion		Med
CD4	Human	M-T477	Qdot 605	T cell Lineage	T cell, T memory	High
CD19	Human	HIB19	Pacific Blue	exclusion		Low
Biotin	Human	Streptavidin	Qdot 655	secondary reagent to PD-1	T cell	Low
CD28	Human	CD28.2	PE-Cy5	memory/differentiation	T cell, T memory	Med
IFN-gamma	Human	B27	APC	function	T cell, T helper	High
CD45RO	Human	UCHL1	Qdot 545	memory/differentiation	T cell, T memory	High
CD8	Human	RPA-T8	Qdot 585	T cell Lineage	T cell	High
PD-1	Human	MIH4	Biotin	memory/differentiation	T cell	Low
CD57	Human	NK-1	Qdot 705	memory/differentiation	T cell	High
CD27	Human	M-T271	PE-Cy7	memory/differentiation	T cell, T memory	Med
ccr7	Human	150503	Alexa Fluor 680	memory/differentiation	Test Group	Low
Dead cells	Human		Live/Dead Fix Violet	viability		Unspecified
CD3	Human	SK7	APC-Cy7	T cell Lineage	T memory, T cell, T helper	High
TNF alpha	Human	MAb11	Alexa Fluor 594	function	T cell, T helper	Med
CD127	Human	R34.34	PE	memory/differentiation	T cell, T memory	Med
IL-2	Human	MQ1-17H12	Alexa Fluor 488	function	T cell, T helper	Med

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OMIP-083

A 21-marker 18-color flow cytometry panel for in-depth phenotyping of human peripheral monocytes

Kathryn E. Hally, Laura Ferrer-Font, Katherine R. Pilkington, Peter D. Larsen

OMIP-082

A 25-color phenotyping to define human innate lymphoid cells, natural killer cells, mucosal-associated invariant T cells, and γδ T cells from freshly isolated human intestinal tissue

Chloe M. Doyle, Nicole L. Fewings, Grahame Ctercteko, Scott N. Byrne, Andrew N. Harman, Kirstie M. Bertram

OMIP-081

A new 21-monoclonal antibody 10-color panel for diagnostic polychromatic immunophenotyping

Erik H. L. P. G. Huys,Willemijn Hobo,Frank W. M. B. Preijers

OMIP-080

29-Color flow cytometry panel for comprehensive evaluation of NK and T cells reconstitution after hematopoietic stem cells transplantation

Sarka Vanikova,Abhishek Koladiya,Jan Musil

OMIP-079

Cell cycle of CD4+ and CD8+ naïve/memory T cell subsets, and of Treg cells from mouse spleen

Ambra Natalini,Sonia Simonetti,Gabriele Favaretto,Giovanna Peruzzi,Fabrizio Antonangeli,Angela Santoni,Miguel Muñoz-Ruiz,Adrian Hayday,Francesca Di Rosa

OMIP-078

A 31-parameter panel for comprehensive immunophenotyping of multiple immune cells in human peripheral blood mononuclear cells

Takuto Nogimori, Yuko Sugawara, Masaya Higashiguchi, Hirotomo Murakami, Hirofumi Akita, Shokichi Takahama, Satoshi Tanaka, Takuya Yamamoto

OMIP-077

Definition of all principal human leukocyte populations using a broadly applicable 14-color panel

Maximilian Boesch, Martina Sykora, Silvia Gasteiger, Florent Baty, Martin H. Brutsche, Sieghart Sopper

OMIP-076

High-dimensional immunophenotyping of murine T-cell, B-cell, and antibody secreting cell subsets

Kyle T. Mincham, Jacob D. Young, Deborah H. Strickland

OMIP-075

A 22-color panel for the measurement of antigen-specific T-cell responses in human and nonhuman primates

Takuto Nogimori, Eiko Moriishi, Mami Ikeda, Shokichi Takahama, Takuya Yamamoto

OMIP-074

Phenotypic analysis of IgG and IgA subclasses on human B cells

Leonard Nettey, Reid Ballard, Thomas Liechti, Rosemarie D. Mason

OMIP-073

Analysis of human thymocyte development with a 14-color flow cytometry panel

Sarah-Jolan Bremer, Laura Glau, Christina Gehbauer, Annika Boxnick, Daniel Biermann, Jörg Siegmar Sachweh, Eva Tolosa, Anna Gieras

OMIP-072

A 15-color panel for immunophenotypic identification, quantification, and characterization of leukemic stem cells in children with acute myeloid leukemia

Marianne A. Petersen, Marie Bill, Carina A. Rosenberg

OMIP-071

A 31-Parameter Flow Cytometry Panel for In-Depth Immunophenotyping of Human T-Cell Subsets Using Surface Markers

Song-Rong Wang, Na Zhong, Xin-Mei Zhang, Zhi-Bin Zhao, Robert Balderas, Liang Li, Zhe-Xiong Lian

OMIP-070

NKp46‐Based 27‐Color Phenotyping to Define Natural Killer Cells Isolated From Human Tumor Tissues

Marie Frutoso, Florian Mair, Martin Prlic

OMIP-069

Forty‐Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood

Lily M. Park, Joanne Lannigan, Maria C. Jaimes

OMIP-068

High‐Dimensional Characterization of Global and Antigen‐Specific B Cells in Chronic Infection

Katherine Cascino, Mario Roederer, Thomas Liechti

OMIP-067

28‐Color Flow Cytometry Panel to Evaluate Human T‐Cell Phenotype and Function

Phillip A. Swanson II, Robert A. Seder

OMIP-066

Identification of Novel Subpopulations of Human Group 2 Innate Lymphoid Cells in Peripheral Blood

Yoichiro Ohne

OMIP-065

Dog Immunophenotyping and T‐Cell Activity Evaluation with a 14‐Color Panel

Stanislav Pantelyushin, Elisabeth Ranninger, Regula Bettschart‐Wolfensberger, Johannes vom Berg

OMIP-064

A 27‐Color Flow Cytometry Panel to Detect and Characterize Human NK Cells and Other Innate Lymphoid Cell Subsets, MAIT Cells, and γδ T Cells

Nina Hertoghs, Katharine V. Schwedhelm, Kenneth D. Stuart, Margaret Juliana McElrath, Stephen C. De Rosa

OMIP-063

28‐Color Flow Cytometry Panel for Broad Human Immunophenotyping

Kathryn Payne, Wenyan Li, Robert Salomon, Cindy S. Ma

OMIP-062

A 14‐Color, 16‐Antibody Panel for Immunophenotyping Human Innate Lymphoid, Myeloid and T Cells in Small Volumes of Whole Blood and Pediatric Airway Samples

Dawid Swieboda, Yanping Guo, Sophie Sagawe, Ryan S. Thwaites, Simon Nadel, Peter J.M. Openshaw, Fiona J. Culley

OMIP-061

20‐Color Flow Cytometry Panel for High‐Dimensional Characterization of Murine Antigen‐Presenting Cells

Anthony T. DiPiazza, Juliane P. Hill, Barney S. Graham, Tracy J. Ruckwardt

OMIP-060

30‐Parameter Flow Cytometry Panel to Assess T Cell Effector Functions and Regulatory T Cells

Thomas Liechti, Mario Roederer

OMIP-059

Identification of Mouse Hematopoietic Stem and Progenitor Cells with Simultaneous Detection of CD45.1/2 and Controllable Green Fluorescent Protein Expression by a Single Staining Panel

Marcus Eich, Andreas Trumpp, Steffen Schmitt

OMIP-058