< OMIPS Directory

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).


MACHINE: Bio-Rad Ze5

Marker Target Clone Fluorochrome Purpose
Dead cells Human Live/Dead Fix Blue viability
CD14 Human MfP9 BUV395 monocyte
CD16 Human 3G8 BUV496 monocyte
HLA-DR Human G46-6 BUV661 DC
CD56 Human NCAM16.2 BUV737 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
CCR10 Human 314305 APC Th subset
CD123 Human 32703 Alexa Fluor 700 pDC (plasmacytoid DC)
CD127 Human RDR5 APC-eFluor 780 Treg