FIGURE 8.

Human cell immunophenotyping and reactivity with CD47/SIRPα antagonists via flow cytometry. A, gating strategy to identify B cell, CD4-T cell, CD8-T cell, and NK cell subpopulations. B cells = CD19⁺CD20⁺; CD4 T cells = CD3⁺CD4⁺; CD8 T cells = CD3⁺CD8⁺; NK cells = CD3⁻CD14⁻CD19⁻CD16⁺CD56⁺. B, gating strategy to identify monocytes. Monocytes = FSC^hiCD14⁺. C, gating strategy to identify neutrophils, eosinophils, and basophils. Neutrophils = CD3⁻CD14⁻CD19⁻CD56⁻SSC^hiCCR3⁻; eosinophils = CD3⁻CD14⁻CD19⁻CD56⁻SSC^hiCCR3⁺; basophils = CD3⁻CD14⁻CD19⁻CD56⁻SSC^loCD123^hiCCR3⁺. D, gating strategy to identify red blood cells. RBC = CD235a⁺. E, heat map representation of percentage of human peripheral blood cells positively stained with the indicated agent/clone targeting either SIRPα (N3612, 15-414, SE5A5, and REA144) or CD47 (CV1). FMO, fluorescence minus one. N3612 and CV1 proteins were used at 250 nm, and the anti-SIRPα antibodies were used at 1:50 dilution.