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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

Marker Target Clone Fluorochrome Purpose
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
CD56 Human PE-Cy7 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