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

PURPOSE: 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.


MACHINE: Cytek Aurora 5 Laser

Marker Target Clone Fluorochrome Purpose
Dead cells Human Live/Dead Fix Blue Viability
CD45 Human 2D1 PerCP Leukocyte
CD3 Human SK7 Brilliant Violet 510 Pan T cell, NKT cells
CD4 Human SK3 cFluor YG584 CD4 T and NKT cells
CD8 Human SK1 BUV805 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 BUV496 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 BUV661 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 BB515 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 BUV737 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 BUV395 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 BUV563 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 BUV615 NK cell differentiation