OMIP-001 Paper

Quality and phenotype of Ag-responsive human T-cells

Yolanda D. Mahnke, Mario Roederer Cytometry PART A, Volumn 77A, Issue 9, 819-820 (2010)

PURPOSE: 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.

CELL TYPE: Peripheral Blood Mononuclear Cells

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
IFN-gamma Human B27 APC Function
IL-2 Human MQ1-17H12 Alexa Fluor 488 Function
TNF alpha Human MAb11 Alexa Fluor 594 Function
CD3 Human SK7 APC-Cy7 Lineage
CD4 Human M-T477 Qdot 605 Lineage
CD8 Human RPA-78 Qdot 585 Lineage
CCR7 Human 150503 Alexa Fluor 680 Memory/differentiation
CD27 Human M-T271 PE-Cy7 Memory/differentiation
CD28 Human CD28.2 PE-Cy5 Memory/differentiation
CD45RO Human UCHL1 Qdot 545 Memory/differentiation
CD57 Human NK-1 Qdot 705 Memory/differentiation
CD127 Human R34.34 PE Memory/differentiation
PD-1 Human MIH4 Biotin Memory/differentiation
Biotin SAV Qdot 655 Memory/differentiation
CD14 Human M5E2 Pacific Blue Dump
CD19 Human HIB19 Pacific Blue Dump
Viability Dye Live/Dead Fix Violet Dump
OMIP-002 Paper

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

Pratip K Chattopadhyay, Mario Roederer, David A. Price Cytometry PART A, Volume 77A, Issue 9, 821-822 (2010)

PURPOSE: 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.1, 21

CELL TYPE: Fresh or cryopreserved PBMC, bone marrow mononuclear cells

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
CD3 Human SK7 APC-Cy7 T-cell subset
CD4 Human MT-477 Qdot 705 T-cell subset
CD8 Human RPA-T8 PE-Alexa 594 T-cell subset
Multimer 1 PE Ag-specificity
Multimer 2 Qdot 565 Ag-specificity
Multimer 3 Qdot 605 Ag-specificity
Multimer 4 Qdot 800 Ag-specificity
CD45RO Human UCHL1 APC-Alexa 700 Maturity
CCR7 Human 150503 PE-Alexa 750 Maturity
CD27 Human 1A4 PE-Cy5 Maturity
CD127 Human R34.34 PE-Alexa 700 Maturity
PD-1 Human MIH4 Biotin Maturity
Biotin SAV APC Maturity
CD57 Human NK-1 FITC Maturity
CD14 Human M5E2 Pacific Blue Dump
CD19 Human HIB19 Pacific Blue Dump
Viability Dye Live/Dead Fix Violet Dump
OMIP-003 Paper

Phenotypic analysis of human memory B cells

Chungwen Wei, John Jung, Inaki Sanz Cytometry PART A, Volume 79A, Issue 11, 894-896 (2011)

PURPOSE: 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.

CELL TYPE: Fresh or cryopreserved PBMC, bone marrow, and tonsil mononuclear cells

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
IgD Human IA6-2 FITC Differentiation
CXCR3 Human 1C6/CXCR3 PE Homing
CD24 Human SN3 PE-Alexa 610 Differentiation
CD21 Human B-ly4 PE-Cy5 Activation
CD38 Human HIT2 PerCP-Cy5.5 Differentiation
CD45R (B220) Human RA3/6B2 PE-Cy7 Differentiation
CD3 Human SP-34-2 Pacific Blue Exclusion
Viability Dye Live/Dead Fix Aqua Exclusion
CD27 Human CLB-27/1 Qdot 605 Differentiation
CD95 Human DX2 APC Activation
VH4-34- encoded idiotype 9G4 Biotin Autoreactive
Biotin SAV Alexa Fluor 680 Autoreactive
CD19 Human SJ25C1 APC-Cy7 Lineage
OMIP-004 Paper

In-depth characterization of human T regulatory cells

Angelique Biancotto, Pradeep K. Dagur, J. Chris Fuchs, Marc Langweiler, J. Philip McCoy Jr Cytometry PART A, Volume 81A, Issue 1, 15-16 (2012)

PURPOSE: 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.2

CELL TYPE: Fresh or cryopreserved PBMCs, ACK-lysed blood

Panel: Markers Target Clone Fluorochrome Purpose
Viability Live/Dead Fix Aqua Viability
CD45 Human HI30 Qdot 800 Leukocyte Gating
CD3 Human sk7 APC-Cy7 T-Cell
CD4 Human RPA-T4 V450 Helper Subset
CD8 Human 3B4 Qdot 605 Suppressor Subset
CD25 Human M-A251 PE-Cy7 Treg Gating
CD27 Human CLB-27/1 Qdot 655 Treg Gating
CD38 Human HIT2 PerCP-Cy5.5 Treg Gating
CD39 Human A1 Alexa Fluor 488 Suppressor Subset
CD45RA Human 2H4LDH11LDB9 PE-Texas Red Naiive
CD103 Human LF61 PE-Cy5 Naiive
CD127 Human HIL-7R-M21 Alexa Fluor 647 Treg Gating
CD197 Human 150503 Alexa Fluro 700 Treg Gating
HLR DR Human Tu96 PE-Cy5.5 Treg Gating
FoxP3 Human PCH101 PE Treg Marker
OMIP-005 Paper

Quality and phenotype of antigen-responsive rhesus macaque T cells

Kathryn E. Foulds, Mitzi Donaldson, Mario Roederer Cytometry PART A, Volume 81A, Issue 5, 360-361 (2012)

PURPOSE: 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 TYPE: Peripheral Blood Mononuclear Cells

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
Viability Zombie Aqua Exclusion
CD3 Rhesus macaque SP34.2 APC-Cy7 T-Cells
CD4 Rhesus macaque S3.5 Qdot 605 T-Cells
CD8 Rhesus macaque RPA-T4 Pacific Blue T-Cells
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
CD45RA Rhesus macaque L48 PE-Cy7 Memory
CD28 Rhesus macaque 28.2 PE-Cy5 Memory
CCR7 Rhesus macaque 150503 Alexa Fluor 680 Memory
OMIP-006 Paper

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

David M. Murdoch, Janet S. Staats, Kent J. Weinhold Cytometry PART A, Volume 81A, Issue 4, 281-283 (2012)

PURPOSE: 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 TYPE: Cryoperserved Peripheral Blood Mononuclear Cells

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
CD3 Human UCHT1 V500 T-Cell
CD4 Human SK3 PerCP-Cy5.5 T-Cell
CD45RO Human UCHL1 FITC Maturation/Differentiation
CD25 Human B1.49.9 ECD Activation/Identification
FoxP3 Human PCH101 PE Treg Identification
Helios Human 22F6 Alexa Fluor 647 Treg Subset
CD39 Human eBioA1 PE-Cy7 Treg Subset
CD49d Human 9F10 PE-Cy5 Treg Subset
Viability Live/Dead Fix Violet Exclusion
OMIP-007 Paper

Phenotypic analysis of human natural killer cells

Micheal A. Eller, Jeffrey R. Currier Cytometry PART A, Volume 81A, Issue 6, 447-449 (2012)

"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 TYPE: Fresh and Cryoperserved Peripheral Blood Mononuclear Cells

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
Viability Live/Dead Fix Violet Viability
CD3 Human S4.1 PE-Texas Red Dump
CD4 Human SFCI12T4D11 ECD Dump
CD14 Human TuK4 PE-Cy5 Dump
CD19 Human SJ25-C1 PE-Cy5 Dump
CD16 Human 3G8 Pacific Blue NK Subset
CD56 Human NCAM16.2 PE-Cy7 NK-Subset
CD8 Human SK1 APC-H7 NK-Subset
Integrin alpha 4 beta 7 Human ACT-1 Qdot 655 Homing
CD62L Human DREG56 Qdot 605 Homing
CD158a/h Human HP-MA4 PerCP-Cy5.5 KIR Receptors
CD158b1/b2/j Human DX27 PE KIR Receptors
CD158e1 Human DX9 Alexa Fluor 700 KIR Receptors
HLA DR Human G46-6 FITC Activation
CD57 Human HCD57 APC Differentiation
OMIP-008 Paper

Measurement of Th1 and Th2 cytokine polyfunctionality of human T cells

Cindy L. Zuleger, Mark R. Albertini Cytometry PART A, Volume 81A, Issue 6, 450-452 (2012)

PURPOSE: 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 TYPE: Peripheral Blood Mononuclear Cells, T cell clones/lines, tumor-resident T cell lines

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
IL-2 Human MQ1-17H12 PerCP-Cy5.5 Function
IL-4 Human 8D4-8 Alexa Fluor 488 Fuction
IL-10 Human JES3-9D7 PE Function
IFN-gamma Human B27 APC Function
TNF alpha Human MAb11 PE-Cy7 Function
CD3 Human UCHT1 Alexa Fluor 700 Lineage
CD4 Human RPA-T4 APC-Cy7 Lineage
CD8 Human 3B5 Qdot 605 Lineage
CD14 Human M5E2 Pacific Blue Dump
CD19 Human HIB19 eFluor 450 Dump
Viability Dye Live/Dead Fix Violet Dump
OMIP-009 Paper

Characterization of antigen-specific human T-cells

Laurie Lamoreaux, Richard A. Koup, Mario Roederer Cytometry PART A, Volume 81A, Issue 5, 363-363 (2012)

PURPOSE: 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 TYPE: Cryopreserved or Fresh Peripheral Blood Mononuclear Cells

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
IFN-gamma Human B27 APC Function
IL-2 Human MQ1-17H12 PE Fuction
TNF alpha Human Mab11 FITC Function
CD3 Human SP342 APC-Cy7 Lineage
CD4 Human T4 ECD Lineage
CD8 Human RPA-T4 Pacific Blue Lineage
CD45RA Human L48 PE-Cy7 Memory/Differentiation
CD28 Human CD28.2 PE-Cy5 Memory/Differentiation
CCR7 Human 150503 Alexa Fluor 680 Memory/Differentiation
Viability Dye Live/Dead Fix Aqua Viability
OMIP-010 Paper

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

Frank W. M. B. Preijers, Erik Huys, Bijan Moshaver Cytometry PART A, Volume 81A, Issue 6, 453-455 (2012)

PURPOSE: 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.1, 21

CELL TYPE: Fresh Cerebral Spinal Fluids, Bone Marrow, Peripheral Blood

MACHINE: Beckman Coulter Navios

Panel: Markers Target Clone Fluorochrome
CD34 Human 581 FITC
Kappa Light Chain Human Polyclonal/Rabbit FITC
CD7 Human 8H8.1 PE
Lambda Light Chain Human Polyclonal/Rabbit PE
CD10 Human ALB1 ECD
CD4 Human T4 PE-Cy5.5
CD56 Human NKH-1 PE-Cy7
CD117 Human 4G7 PE-Cy7
CD15 Human 80H5 Pacific Blue
CD20 Human HRC20 Pacific Blue
CD45 Human J33 Krome Orange
CD3 Human UCHT1 APC
CD33 Human D3HL60.251 APC
CD8 Human T8 APC-Alexa 700
CD19 Human J4.119 APC-Alexa 750
OMIP-011 Paper

Characterization of circulating endothelial cells (CECs) in peripheral blood

Raskit Lachmann, Paola Lanuti, Sebastiano Miscia Cytometry PART A, Volume 81A, Issue 7, 549-551 (2012)

PURPOSE: 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 TYPE: Whole blood, RBC-lysed and washed

MACHINE: BD FACSCanto 2

Panel: Markers Target Clone Fluorochrome Purpose
Viability Dye Nile Red Viability
CD45 Human HI30 Alexa Fluor 700 Exclusion of Leucocytes
DNA Syto 16 DNA marker
CD31 Human M89D3 Alexa Fluor 647
CD34 Human 581 ECD Endothelial cell marker
CD146 Human P1H12 PE
CD117 Human 104D2 PE-Cy7 Progenitor marker
CD106 Human 51-10C9 PE-Cy5 Activation marker
OMIP-012 Paper

Phenotypic and numeric determination of human leukocyte reconstitution in humanized mice

Brian R. Long, Cheryl A. Stoddart Cytometry PART A, Volume 81A, Issue 8, 646-648 (2012)

PURPOSE: 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). 11, 2

CELL TYPE: Whole blood, Ficoll-separated PBMC, splenocytes

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
CD45 Mouse 30-F11 APC Exclusion of mouse leukocytes
CD45 Human HI30 Alexa Fluor 700 Human leukocyte detection
CD3 Human UCHT1 ECD Human T Cell detection
CD19 Human HIB19 APC-Cy7
CD56 Human HCD56 PE-Cy7 Human NK cell detection
CD4 Human RPA-T4 Pacific Blue Human CD4+ T cell subset
CD8 Human 3B5 Qdot 605 Human CD8+ T cell subset
HLA DR Human L243 FITC Activation
CD38 Human HIT2 PE Activation
Isotype Control Human MOPC-21 PE Set gate for CD38 expression
OMIP-013 Paper

Differentiation of human T-cells

Yolanda D. Mahnke, Margaret H. Beddall, Mario Roederer Cytometry PART A, Volume 81A, Issue 11, 935-936 (2012)

PURPOSE: 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 TYPE: PBMC

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
CD3 Human SK7 APC-H7 Lineage
CD4 Human M-T477 Qdot 605 Lineage
CD8 Human RPA-T8 Qdot 585 Lineage
CCR7 Human 150503 Alexa Fluor 680 Memory/Differentiation
CD27 Human O323 FITC Memory/Differentiation
CD28 Human CD28.2 PE-Cy5 Memory/Differentiation
CD31 Human WM59 PE-Cy7 Memory/Differentiation
CD45RA Human HI100 APC Memory/Differentiation
CD57 Human NK-1 Qdot 705 Memory/Differentiation
CD95 Human DX2 PE Memory/Differentiation
CD127 Human A019D5 Brilliant Violet 421 Memory/Differentiation
CD244 Human C1.7 PE-Cy5.5 Memory/Differentiation
Viability Dye Live/Dead Fix Aqua Dump
OMIP-014 Paper

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

Stephen C. De Rosa, Donald K. Carter, M. Juliana McElrath Cytometry PART A, Volume 81A, Issue 5, 362-363 (2012)

PURPOSE: 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 TYPE: Cryopreserved PBMC

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
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-Texa Red Lineage
CD4 Human 13B8.2 APC-Alex 750 Lineage
CD8 Human SK1 PerCP-Cy5.5 Lineage
CD14 Human TuK4 Qd 655 Dump
Viability Dye Live/Dead Fix Aqua Viability
OMIP-015 Paper

Human regulatory and activated T-cells without intracellular staining

Yolanda D. Mahnke, Margaret H. Beddall, Mario Roederer Cytometry PART A, Volume 83A, Issue 2, 179-181 (2013)

PURPOSE: 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 TYPE: PBMC

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
CD3 Human OKT3 Brilliant Violet 785 Lineage
CD4 Human OKT4 Qdot 605 Lineage
CD8 Human RPA-T8 Qdot 585 Lineage
CD25 Human M-A251 PE-Cy5 T-reg
CD127 Human eBioRDR5 APC-eFluor 780 T-reg
CD39 Human eBIOA1 PE-Cy7 T-reg Functional
CD73 Human AD2 PE T-reg Functional
CD38 Human HIT2 PE-CF594 Activation/Differentiation
CD45RA Human 5H9 Qdot 705 Activation/Differentiation
CD45RO Human UCHL1 FITC Activation/Differentiation
HLA DR Human G46-4 Alexa Fluor 680 Activation/Differentiation
PD-1 Human EH12.2H7 Brilliant Violet 421 Activation/Differentiation
Viability Dye Live/Dead Fix Aqua Viability
OMIP-016 Paper

Characterization of antigen-responsive macaque and human T-cells

Sabrina Guenounou, Nathalie Bosquet, Claudia J. Dembek, Roger Le Grand, Antonio Cosma Cytometry PART A, Volume 83A, Issue 2, 182-184 (2013)

PURPOSE: 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 TYPE: Fresh or cryopreserved PBMCs

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
Viability Dye Live/Dead Fix Blue Viability
CD3 Cynomolgus/Human SP34-2 APC-Cy7 Lineage
CD4 Cynomolgus/Human L200 PerCP-Cy5.5 Lineage
CD8 Cynomolgus/Human RPA-T8 V500 Lineage
CD45RA Cynomolgus/Human L48 PE-Cy7 Memory/differentiation
CD154 Cynomolgus/Human TRAP1 FITC CD4 Activation
MIP-1 beta Cynomolgus/Human D21-1351 PE Function
IFN gamma Cynomolgus/Human B27 V450 Function
TNF alpha Cynomolgus/Human MAb11 Alexa Fluor 700 Function
IL-2 Cynomolgus/Human MQ1-17H12 APC Function
OMIP-017 Paper

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

Yolanda D. Mahnke, Margaret H. Beddall, Mario Roederer Cytometry PART A, Volume 83A, Issue 5, 439-440 (2013)

PURPOSE: 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 TYPE: PBMC

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
CD3 Human UCHT1 Alexa Fluor 594 Lineage
CD4 Human OKT4 Qdot 800 Lineage
CD8 Human RPA-T8 Qdot 585 Lineage
CXCR5 Human RF8B2 Alexa Fluor 647 T-FH
CCR4 Human TG6/CCR4 PE-Cy7 Th subset
CCR6 Human G034E3 Brilliant Violet 605 Th subset
CCR10 Human 6588-5 PE Th subset
CXCR3 Human 1C6/CXCR3 PE-Cy5 Th subset
CCR7 Human 150503 Alexa Fluor 680 Differentiation
CD45RA Human 5H9 Qdot 655 Differentiation
CD161 Human DX12 FITC Exploratory
PD-1 Human EH12.2H7 Brilliant Violet 421 Exploratory
Viability Dye Live/Dead Fix Aqua Dump
OMIP-018 Paper

Chemokine receptor expression on human T helper cells

Tess Brodie, Elena Brenna, Federica Sallusto Cytometry PART A, Volume 83A, Issue 6, 530-532 (2013)

PURPOSE: 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 TYPE: Fresh PBMC

MACHINE: BD LSR Fortessa

Panel: Markers Target Clone Fluorochrome Purpose
CD3 Human S4.1 PE-Cy5.5 T-Cell Subset
CD4 Human S3.5 PE-Texas Red T-Cell Subset
CD45RA Human MEM-56 Qdot 655 Maturity
CCR7 Human G043H7 Brilliant Violet 421 Maturity
CD95 Human DX2 PerCP-eFluor 710 Maturity
CCR4 Human 1G1 PE-Cy7 Polarization
CXCR3 Human 1C6/CXCR3 PE-Cy5 Polarization
CCR6 Human 11A9 Biotin Polarization
Streptavidin Human Qdot 800 Polarization
CXCR5 Human 51505 Qdot 605 Polarization
CCR5 Human 2D7/CCR5 APC-Cy7 Polarization
CCR3 Human 61828 rat APC Polarization
CCR10 Human 314305 rat PE Polarization
CRTh2 Human BM16 rat FITC Polarization
CD14 Human M5E2 V500 Exclusion
CD16 Human 3G8 V500 Exclusion
CD19 Human HIB19 V500 Exclusion
Viability Dye Live/Dead Fix Aqua Exclusion
OMIP-019 Paper

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

Yolanda D. Mahnke, Margaret H. Beddall, Mario Roederer Cytometry PART A, Volume 83A, Issue 8, 676-678 (2013)

PURPOSE: 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 TYPE: PBMC

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
CD3 Human OKT3 Brilliant Violet 785 Lineage
CD1d /PBS-57 multimer Human N/A PE iNKT
TCR-DV1 Human TS8.2 FITC γδT-cells
TCR-DV2 Human B6 Alexa Fluor 594 γδT-cells
TCR-GV9 Human B3 APC γδT-cells
CD34 Human HI100 Brilliant Violet 421 Hematopoietic stem cells
CCR5 Human 2D7/CCR5 APC-Cy7 Phenotyping
CCR7 Human 150503 Alexa Fluor 680 Phenotyping
CD4 Human OKT4 Qdot 605 Phenotyping
CD8 Human RPA-T8 Qdot 585 Phenotyping
CD27 Human 1A4LDG Qdot 655 Phenotyping
CD28 Human CD28.2 PE-Cy5 Phenotyping
CD45RA Human MEM-56 PE-Cy5.5 Phenotyping
Viability Dye Human Live/Dead Fix Blue Dump
OMIP-020 Paper

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

Kilian Wistuba-Hamprecht, Graham Pawelec, Evelyna Derhovanessian Cytometry PART A, Volume 85, Issue 6, 522-524 (2013)

PURPOSE: 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 TYPE: PBMC

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome
CD3 Human UCHT-1 Alexa Fluor 700
CD4 Human OKT4 PE-Cy7
CD8 Human SK1 APC-H7
CD16 Human 3G8 Brilliant Violet 711
CD27 Human 0323 APC
CD28 Human CD28.2 PE
CD45RA Human H100 Pacific Blue
TCR gamma/delta Human 11F2 Purfied
TCR V delta 1 Human TS8.2 FITC
TCR V delta 2 Human B6 PerCP
F(ab′)2-Fragment goat Mouse RPA-T8 Pacific Orange
Viability Dye EMA
OMIP-021 Paper

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

Nicholas A. Gherardin, David S. Ritchie, Dale I. Godfrey, Paul J. Neeson Cytometry PART A, Volume 85, Issue 7, 573-575 (2014)

PURPOSE: 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 TYPE: PBMC

MACHINE: BD LSR Fortessa

Panel: Markers Target Clone Fluorochrome Purpose
Viability Dye Live/Dead Fix Blue 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 Subsets
CD28 Human 28.2 PE-Cy5 Memory T-cell Subsets
CD45RA Human HI100 PerCP-Cy5.5 Memory T-cell Subsets
CD45RO Human UCHL1 Alexa Fluor 700 Memory T-cell Subsets
CCR7 Human 3D12 PE-Cy7 Memory T-cell Subsets
hCD1d-PBS44 Brilliant Violet 421 NKT cells
TCR delta/gamma Human 11F2 FITC γδ T-cells
TCR V delta 1/TCR V delta 2 Human 3C10 PE MAIT cells
CD161 Human HP-3G10 Brilliant Violet 605 MAIT cells
OMIP-022 Paper

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

Andrew J. Graves, Marcelino G. Padilla, David A. Hokey Cytometry PART A, Volume 85, Issue 7, 576-579 (2014)

PURPOSE: 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 TYPE: Cryopreserved PBMC (adult and infant)

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
Viability Dye Live/Dead Fix Blue Dump
CD14 Human M5E2 V500 Dump
CD19 Human HIB19 V500 Dump
CD3 Human UCHT1 ECD Phenotyping
CD4 Human RPA-T4 APC-eFluor 780 Phenotyping
CD8 Human HIT8a Alexa Fluor 700 Phenotyping
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 Th1
TNF alpha Human MAb11 PE-Cy7 Th1
IL-17A/td> Human BL168 PerCP-Cy5.5 Th17
IL-22 Human IL22JOP APC Th22
CD107a Human H4A3 Alexa Fluor 488 Degranulation
CD154 Human TRAP1 PE-Cy5 Activation/B-Cell Help
OMIP-023 Paper

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

Jozsef Bocsi, Susanne Meizer, Ingo Dahnert, Attila Tarnok Cytometry PART A, Volume 85, Issue 9, 781-784 (2014)

PURPOSE: 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 TYPE: Fresh EDTA-treated peripheral blood, RBC-lysed and Cord-blood

MACHINE: Beckman Coulter Navios

Panel: Markers Target Clone Fluorochrome Purpose
CD45 Human J.33 Pacific Blue Pan-leukocyte antigen
CD3 Human SP34-2 V500 T-cells
CD8 Human B9.11 FITC Cytotoxic T-cells
CD4 Human SK3 APC-H7 T-helper cells
CD25 Human B1.49.9 ECD IL-2 Receptor α
CD127 Human R 34.34 APC-Alexa 700 IL-7 Receptor α
CD19 Human J3–119 FITC B-cells
CD38 Human LS198.4.3 PE-Cy5.5 Activated T and B-cells
HLA DR Human Immu-357 APC MHC-II
CD16 Human 3G8 PE-Cy7 Fcγ Rec III
CD56 Human N901(NKH-1) PE-Cy7 N-Cam
CD14 Human RMO52 FITC LPS co-receptor
CD69 Human TP1.55.3 PE Early activation
OMIP-024 Paper

Pan-leukocyte immunophenotypic characterization of PBMC subsets in human samples

Gemma Moncunill, Hannah Han, Carlota Dobano, M. Juliana McElrath, Stephen C. De Rosa Cytometry PART A, Volume 85, Issue 12, 995-998 (2014)

PURPOSE: 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 TYPE: Cryopreserved PBMC from adults and infants

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
CD3 Human UCHT1 ECD Lineage T cells
CD4 Human SK3 BUV395 Lineage T cells
CD8 Human SK1 PerCP-Cy5.5 Lineage T cells
CD19 Human SJ25C1 BUV737 B cells
CD14 Human MφP9 Brilliant Violet 711 Monocytes
CD56 Human HCD56 Brilliant Violet 605 NK cells and NK T-like cells
CD16 Human 3G8 APC-Cy7 NK cells and monocytes
TCR delta/gamma Human 11F2 PE-Cy7 γδ T cells
TCR V delta 2 Human B6 PE γδ T cells
CD25 Human M-A251 Brilliant Violet 421 Tregs
CD127 Human AO19D5 APC Tregs/memory/differentiation
CD45RA Human HI100 Brilliant Violet 650 Memory/differentiation
CCR7 Human G043H7 Brilliant Violet 785 Memory/differentiation
CD57 Human NK-1 FITC Memory/differentiation
HLA DR Human L243 Brilliant Violet 570 Activation
CD38 Human HIT2 PE-Cy5 Activation/plasmablasts
NKG2C Human 134591 Alexa Fluor 700 NK receptor
Viability Dye Live/Dead Fix Blue Viability
OMIP-025 Paper

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

Gemma Moncunill, Carlota Dobano, M. Juliana McElrath, Stephen C. Rosa Cytometry PART A, Volume 87, Issue 4, 289-292 (2015)

PURPOSE: 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 TYPE: Cryopreserved PBMC

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
CD3 Human UCHT1 Brilliant Violet 570 T lineages
CD8 Human RPA-T8 Brilliant Violet 711 T lineages
CD4 Human SK3 BUV395 T lineages
CXCR5 Human MU5UBEE PE-eFluor 610 T-FH
PD-1 Human eBioJ105 PE-Cy7 T-FH
CD45RA Human HI100 APC-H7 Memory/Differentiation
CCR7 Human G043H7 Brilliant Violet 785 Memory/Differentiation
CD56 Human HCD56 Brilliant Violet 650 NK cells, NKT-like cells
IFN-gamma Human B27 V450 Function
IL2 Human MQ1–17H12 PE Function
TNF alpha Human MAb11 FITC Function
IL-4 Human MP4–25D2 PerCP-Cy5.5 Function
IL-21 Human 3A3-N2 APC Function
CD154 Human 24–31 Brilliant Violet 605 Activation
CD14 Human M5E2 Brilliant Violet 510 Dump
Viability Dye Live/Dead Fix Blue Dump
OMIP-026 Paper

Phenotypic analysis of B and plasma cells in rhesus macaques

Berit Neumann, Sieghart Sopper, Christiane Stahl-Hennnig Cytometry PART A, Volume 87, Issue 9, 800-802 (2015)

PURPOSE: 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 TYPE: Fresh whole blood, PBMCs, mononuclear cells of bone marrow, lymph node, spleen, tonsil

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
CD45 Rhesus D058-128 V500 Leukocyte definition
CD10 Rhesus HI10a APC-Cy7 Maturation marker
CD3 Rhesus SP34-2 Alexa Fluor 700 Lineage
CD20 Rhesus L27 PE-Cy7 Lineage
CD21 Rhesus B-Ly4 FITC Differentiation
CD27 Rhesus M-T271 APC Differentiation
CD69 Rhesus TP1.55.3 ECD Activation
CD80 Rhesus L307.4 PE Activation
CD197 Rhesus G043H7 Brilliant Violet 421 Homing
CCR7 Rhesus G043H7 Brilliant Violet 421 Homing
IgD Rhesus MQ1–17H12 PE Ig class switching
CD19 Rhesus J3.119 PE Linege
CD27 Rhesus O323 Brilliant Violet 650 Differentiation
CD38 Rhesus OKT10 APC Differentiation, plasma cells
CD138 Rhesus DL-101 FITC Plasma Cells
CD95 Rhesus DX2 Biotin Activation
CXCR4 Rhesus 12G5 PE-CF594 Homing
Ki67 Rhesus B56 PerCP-Cy5.5 Proliferation
Streptavidin Brilliant Violet 570 Counterstain of Biotin-conjugated CD95 and IgD
OMIP-027 Paper

Functional analysis of human natural killer cells

Margaret C. Costanzo, Matthew Creegan, Kerri G. Lal, Micheal A. Eller Cytometry PART A, Volume 87, Issue 9, 803-805 (2015)

PURPOSE: 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 TYPE: Cryopreserved PBMC

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
Viability Dye Human Live/Dead Fix Aqua Viability
CD3 Human SP34-2 APC-Cy7 Exclusion
CD4 Human SK3 APC-H7 Exclusion
CD14 Human TuK4 PE-Cy5 Exclusion
CD19 Human Sj25-C1 PE-Cy5 Exclusion
CD16 Human 3G8 Pacific Blue NK subsets
CD56 Human NCAM16.2 PE-Cy7 NK subsets
CD107a Human H4A3 FITC Degranulation
IFN-gamma Human 4S.B3 Brilliant Violet 711 Function
TNF alpha Human Mab11 Brilliant Violet 650 Function
Perforin Human B-D48 PE Function
Granzyme B Human GB11 PE-CF594 Function
CD8 Human RPA-T8 Brilliant Violet 785 Function
EOMES Human Dan11mag PerCP-eFluor 710 Transcription factor
CD57 Human HCD57 APC Differentiation
OMIP-028 Paper

Activation panel for Rhesus macaque NK cell subsets

Nicholas Pomplun, Kim L. Weisgrau, David T. Evans, Eva G. Rakasz Cytometry PART A, Volume 87, Issue 10, 890-893 (2015)

PURPOSE: 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 TYPE: Fresh PBMC and lymph node samples

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
Viability Dye Live/Dead Fix NIR Viability
CD45 Rhesus D058-1283 Brilliant Violet 786 hematopoietic cell lineage
CD3 Rhesus SP34-2 Alexa Fluor 700 Exclusion
CD20 Rhesus 2H7 Alexa Fluor 700 Exclusion
CD14 Rhesus M5E2 Alexa Fluor 700 Exclusion
CD8 Rhesus SK1 Brilliant Violet 510 NK subset
CD56 Rhesus B159 FITC NK subset
NKG2A/C/E Rhesus Z199 PE-Cy7 NK subset
KIR3DL01 Rhesus NKFS1 PE NK subset
KIR3DL05 Rhesus GY9 tetramer Brilliant Violet 421 NK subset
NKp46 Rhesus BAB231 PE-Cy5 NK cell differentiation
PD-1 Rhesus EH12.2H7 Brilliant Violet 605 NK cell differentiation
CD69 Rhesus TP1.55.3 ECD Activation
HLA DR Rhesus L243 Brilliant Violet 650 Activation
Ki-67 Rhesus B56 Alexa Fluor 647 Proliferation
OMIP-029 Paper

Human NK-cell phenotypization

Yolanda D. Mahnke, Margaret H. Beddall, Mario Roederer Cytometry PART A, Volume 87, Issue 11, 986-988 (2015)

PURPOSE: 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 TYPE: PBMC

MACHINE: BD LSR2

Panel: Markers Target Clone Fluorochrome Purpose
CD2 Human S5.5 PE-Cy5.5 NK cells
CD16 Human 3G8 Brilliant Violet 421 NK cells
CD56 Human HCD56 Brilliant Violet 570 NK cells
CD3 Human SK7 APC H7 non-NK cells
CD4 Human OKT4 Brilliant Violet 605 non-NK cells
CD158a Human HP-MA4 FITC NK receptors
CD158b Human DX27 PE NK receptors
CD314 Human 1D11 PE-Cy7 NK receptors
CD335 Human BAB281 PE-Cy5 NK receptors
CD337 Human P30-15 Alexa Fluor 647 NK receptors
CD62L Human SK11 Alexa Fluor 680 Differentiation
CCR7 Human 150503 PE-CF594 Differentiation
Viability Dye Live/Dead Fix Aqua Dump
OMIP-030 Paper

Characterization of human T cell subsets via surface markers

Gerhard Wingender, Mitchell Kronenberg Cytometry PART A, Volume 87, Issue 12, 1067–1069 (2015)

PURPOSE: 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 TYPE: Fresh and cryopreserved PBMC

MACHINE:

Panel: Markers Target Clone Fluorochrome Purpose
n/a n/a 2n/a Live/Dead-Blue Viability
CD20 Human 2H7 PE-Cy5.5 B cell exclusion
CD3 Human UCHT1 QD605 Lineage
CD4 Human SK3 BV510/td>
CD8 Human RPA-T8 eF650
CCR7 Human G043H7 Brilliant Violet 421 Naïve and memory subsets
CD45RA Human HI100 BV570 Naïve and memory subsets
CD197 (CCR7) Human 150503 PE-CF594
CD25 Human 2A3 BV711 Treg gating
CD127 Human RDR5 APC-eF780
CCR10 Human 314305 APC CD4+ T cell
CD183 (CXCR3) Human 1C6 FITC Subsets
CD185 (CXCR5) -bio Human RF8B2 -Biotin
+Streptavidin-PE-Cy7 Human n/a PE-Cy7
CD194 (CCR4) Human 1G1 PE
CD196 (CCR6) Human G034 BV786
CD161 Human HP-3G10 PerCP-Cy5.5 mNKT/MAIT cells
CD38 Human HB7 eF450 Activation marker
Ki67 Human B56 AF700 Proliferation
OMIP-031 Paper

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

Satoshi Nemoto, Adam W. Mailloux, Jodi Kroeger, James J. Mulé Cytometry PART A, Volume 89, Issue 5, 427–429 (2016)

PURPOSE: 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 TYPE: Any source containing murine T-cells

MACHINE:

Panel: Markers Target Clone Fluorochrome Purpose
CD3 Mouse 145-2C11 BUV 395 T-cell
CD4 Mouse GK1.5 BUV 805 TH
53-6.7 Mouse SK3 Alexa Fluor 700 TC
CD69 Mouse H1.2F3 PE-CF594 Activation
CD44 Mouse IM7 Alexa Fluor 488 Memory
CD45RA Human 14.8 BV 786 Memory
CD27 Mouse LG.3A10 BV 510 Memory
CD62L Mouse MEL-14 PE-Cy7 Memory
KLRG1 Mouse 2F1/KLRG1 PerCP-Cy5.5 Differentiation
CD127 Mouse SB/199 BUV 737 Differentiation
PD-1 Mouse J43 BV 605 Checkpoint
CTLA4 Mouse UC10-4B9 PE Checkpoint
TIM-3 Mouse B8.2C12 APC Checkpoint
LAG-3 Mouse C9B7W BV 711 Checkpoint
CD45.2 Mouse 104 APC-Cy7 Congenic
DAPI n/a n/a DAPI Viability
OMIP-032 Paper

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

Ashleigh Unsworth, Robin Anderson, Nicole Haynes, Kara Britt Cytometry PART A, Volume 89, Issue 6, 527–530 (2016)

PURPOSE: 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 TYPE: RBC-lysed whole blood, Collagenase IV digested spleen, Collagenase IV digested mammary gland, Collagenase IV digested tumor

MACHINE:

Panel: Laser mAB Clone Fluorochrome Source
Panel 1 405 nm Ly6CMHC II AL-21 M5/114.15.2 BV421 BV711 BD
488 nm CD206 C068C2 FITC Biolegend
561 nm CD11b M1/70 PE BD
Ly6G 1A8 PE-Cy7 BD
640 nm CD11c HL3 APC BD
CD45.2 104 APC-Cy7 eBioscience
Panel 2 405 nm NKp46 29A1.4 BV421 BD
CD62L MEL-14 BV510 BD
CD44 IM7 BV605 BD
CD8 53-6.7 BV711 BD
488 nm CD49b DX5 FITC BD
561 nm CD45.2 104 PE BD
TCRβ H57-597 PE-Cy7 BD
640 nm CD4 GK1.5 APC-Cy7 BD
Both Violet (405 nm) Live/Dead - Fluoro-Gold Sigma Aldrich
Yellow/Green (561nm) Live/Dead - PI Sigma Aldrich
OMIP-033 Paper

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

Tess Brodie, Kristina Rothaeusler, Mireia Sospedra Cytometry PART A, Volume 89, Issue 7, 629-632 (2016)

PURPOSE: 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 TYPE: CSF, Whole Blood-Fresh

MACHINE: BD LSR Fortessa

Panel: Markers Target Clone Fluorochrome Purpose
CD3 Human HITa Alexa Fluor 700 T-cell lineage
CD4 Human S3.5 PE-Texas Red T-cell lineage
CD8 Human SK1 Brilliant Violet 510 T-cell lineage
CD45RA Human HI100 Brilliant Violet 711 Naïve and memory subsets
CCR7 Human G043H7 Brilliant Violet 421 Naïve and memory subsets
CD27 Human O323 APC-Cy7 Naïve and memory subsets
CD28 Human CD28.2 PE-Cy7 Putative autoreactive cells
CCR4 Human L281H4 APC T helper subsets
CCR6 Human G034E3 Brilliant Violet 785 T helper subsets
CRTh2 Human BM16 PE T helper subsets
CD19 Human HIB19 PercP-Cy5.5 B cells
IgD Human IA6-2 Brilliant Violet 605 B cells
CD138 Human MI15 FITC Plasma cells
OMIP-034 Paper

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

Sabine Baumgart, Anette Peddinghaus, Ursula Schulte-Wrede, Henrik E. Mei, Andreas Grützkau Cytometry PART A, Volume 91, Issue 1, 34–38 (2017)

PURPOSE: 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 TYPE: Peripheral blood leukocytes, PBMC

MACHINE:

Specificity Clone Metal isotope Purpose
DNA Intercalator 103 Rh Live/dead cell discrimination
CD45 5B1 141 Pr Pan Leukocytes
CD19 Bu-12 142 Nd B lymphocytes
143 Nd
CD4 RPA-T4 144 Nd T helper lymphocytes
145 Nd
CD45RO UCHL1 146 Nd Memory T lymphocytes
CD20 2H7 147 Sm B lymphocytes
CD560 REA196 148 Nd NK cells
CD25 2A3 149 Sm Activated and regulatory T lymphocytes
CD169 (Siglec-1) 7-239 150 Nd Type I interferon activated monocytes and DC
CD123 6H6 151 Eu Basophils, plasmacytoid DC, myeloid DC
CD45RA 4G11 152 Sm Naive T lymphocytes
CD303 201A 153 Eu plasmacytoid DC
154 Sm
155 Gd
Siglec-8 7C9 156 Gd Eosinophils
157 Gd
CD1c L161 158 Gd Myeloid DC, B cell subset
CD197 (CCR7) G043H7 159 Tb Memory/naïve T lymphocytes subsets
CD11c MJ427G12 160 Gd Monocytes, myeloid DC
161 Dy
CD69 FN50 162 Dy Activated or resident T lymphocytes
163 Dy
IgD IA6-2 164 Dy B lymphocytes subsets
CD127 A019D5 165 Ho Activated and regulatory T lymphocytes
CD36 AC106 166 Er Monocytes and thrombocytes
CD27 L128 167 Er Memory/naïve lymphocytes
CD8 SK1 168 Er Cytotoxic T lymphocytes
CD16 3G8 169 Tm Neutrophils, NK and monocyte subsets
CD3 UCHT1 170 Er T lymphocytes
CD14 TM1 171 Yb Monocytes
CD38 HIT2 172 Yb Activated T and B lymphocytes, plasmablasts, activation, NK cells
173 Yb
HLA-DR L243 174 Yb Dendritic cells, monocytes and B lymphocytes, activation
CD15 W6D3 175 Lu Granulocytes (Neutrophils, Eosinophils)
DNA Intercalator 191/193 Ir Nucleated cells
OMIP-035 Paper

Functional analysis of natural killer cell subsets in macaques

Kim L. Weisgrau, Moritz Ries, Nicholas Pomplun, David T. Evans, Eva G. Rakasz Cytometry PART A, Volume 89, Issue 9, 799–802 (2016)

PURPOSE: 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 TYPE: 721.221 stimulated PBMC

MACHINE:

Specificity Clone Fluorochrome Purpose
Live/dead n/a Near infrared Viability
CD45 D058-1283 BV786 Nonhuman primate specific hematopoietic cell lineage
CD3 SP34-2 PE-CF594 Exclusion
CD20 2H7 PE-CF594
CD8 RPA-T8 BV711 NK subsets
CD16 3G8 Pacific Blue
CD56 B159 PerCP-Cy5.5
NKG2A/C Z199 PE-Cy7
KIR3DL01 NKVFS1 PE
KIR3DL05 GY9 tetramer APC
IFN-γ 4S.B3 FITC NK cell function
TNF-α MAb11 Alexa700
CD107a H4A3 BV605
Granzyme B GB11 BV510
OMIP-036 Paper

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

Zachary R. Healy, David M. Murdoch Cytometry PART A, Volume 89, Issue 10, 889–892 (2016)

PURPOSE: 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 TYPE: PBMC

MACHINE:

Specificity Clone Fluorochrome Purpose
Viability Dye - Zombie Aqua Dump
CD14 M5E2 BV510
CD19 HIB19 BV510
CD3 SK7 BUV395 Phenotype
CD4 SK3 BUV496
CD8 SK1 BUV805
CCR7 G043H7 BV785 Differentiation
CD45RA HI100 BB515
CD28 CD28.2 APC-H7
CD127 A019D5 BV650
KLRG1 SA231A2 AF647
PD-1 EH12.2H7 BV711 Co-inhibitory Receptors
TIM-3 F38-2E2 BV605
LAG-3 3DS223H PE-Cy7
CD160 BY55 PerCP-Cy5.5
BTLA MIH26 BV421
CTLA-4 BNI3 PE-CF594
OMIP-037 Paper

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

Anna C. Belkina, Jennifer E. Snyder-Cappione Cytometry PART A, Volume 91, 175–179 (2016)

PURPOSE: 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 TYPE: Immune cells

MACHINE:

Specificity Fluorochrome Clone Purpose
γδTCR BUV 395 B1 γδ T cells
CD127 BUV 737 HIL-7R-M21 Exhaustion; Treg gating
CD8 BUV 805 SK1 Lineage
PD-1 BV 421 EH12.2H7 Exhaustion
CD3 BV 510 OKT3 T cell lineage
CD16 BV 605 3G8 NK cell lineage/subsets
CD137 BV 650 4B4-1 Activation
CD56 BV 786 NCAM16.2 NK cell lineage/subsets
CD160 AF488 BY55 Exhaustion
Vα24 TCR PE C-15 iNKT lineage
LAG-3 PE-ef610 3DS223H Exhaustion
TIGIT PE-ef710 MBSA43 Exhaustion
TIM-3 PE-Cy7 F38-2E2 Exhaustion
CD1d-PBS57 tetramer APC n/a iNKT lineage
CD4 Af700 RPA-T4 TH cell lineage
CD14 APC-Cy7 HCD14 Monocyte exclusion
CD19 APC-Cy7 HIB19 B cell exclusion
NIR Zombie n/a n/a Viability
CD16/32 n/a n/a FCγRIII blocking
OMIP-038 Paper

Innate immune assessment with a 14 color flow cytometry panel

Kinga K. Smolen, Bing Cai, Tobias R. Kollmann Cytometry PART A, Volume 91, Issue 10, 966–968 (2017)
OMIP-039 Paper

Detection and analysis of human adaptive NKG2C+ natural killer cells

Quirin Hammer, Chiara Romagnani Cytometry PART A, Volume 91, Issue 10, 997–1000 (2017)
OMIP-040 Paper

Optimized gating of human prostate cellular subpopulations

Gervaise H. Henry, Nicolas Loof, Douglas W. Strand Cytometry PART A, Volume 91, Issue 12, 1147–1149 (2017)
OMIP-039 Paper

Detection and analysis of human adaptive NKG2C+ natural killer cells

Quirin Hammer, Chiara Romagnani Cytometry PART A, Volume 91, Issue 10, 997–1000 (2017)
OMIP-041 Paper

Optimized multicolor immunofluorescence panel rat microglial staining protocol

Naama E. Toledano Furman, Karthik S. Prabhakara, Supinder Bedi, Charles S. Cox Jr, Scott D. Olson Cytometry PART A, Volume 93, Issue 2, 182–185 (2017)
OMIP-042 Paper

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

Karl W. Staser, William Eades, Jaebok Choi, Darja Karpova, John F. DiPersio Cytometry PART A, Volume 93, Issue 2, 186–189 (2018)

PURPOSE: 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 TYPE: Human PBMCs

MACHINE:

Specificity Fluorochrome Clone Purpose
CD14 BUV395 MΦP9 monocytes
Live/Dead n/a n/a viability
CD16 BUV496 3G8 monocytes
HLADR BUV661 G46-6 DCs
CD56 BUV737 NCAM 16.2 NKs
CD38 BV421 HIT2 activation
CD20 BV450 L27 B cells
CD4 BV510 SK3 CD4
CD194/CCR4 BV605 L291H4 Th subset
CD8 BV650 RPA-T8 CD8
CD25 BV711 2A3 Treg
CD196/CCR6 BV785 G034 Th subset
CD3 AF488 UCHT1 T cells
CD45RA PerCP-Cy5.5 HI100 naïve/memory
CD183/CXCR3 PE 1C6 Th subset
CD197/CCR7 PE-CF594 150503 central/effector
CD11c PE-Cy5 Bly6 mDCs
CD185/CXCR5 PE-Cy7 RF8B2 Th subset
CCR10 APC 314305 Th subset
CD123 AF700 32703 pDCs
CD127 APC-eF780 RDR5 Treg
OMIP-043 Paper

Identification of human antibody secreting cell subsets

Jeffrey Carrell, Christopher J. Groves Cytometry PART A, Volume 93, Issue 2, 190–193 (2018)
OMIP-044 Paper

28-color immunophenotyping of the human dendritic cell compartment

Florian Mair, Martin Prlic Cytometry PART A, Volume TBD