CD47-SIRPα Binding Assay

Flow-based mouse CD47-Fc (mCD47-Fc) (R&D Systems) binding assay was performed as previously described with some modifications (12). Briefly, spleen cells from indicated mouse strains were blocked with anti-CD16/32 (BD Biosciences) followed by pre-clustering of SIRPα with unconjugated P84 antibody (eBiosciences). Cells were then incubated with serial dilution of mCD47-Fc for 30 minutes followed by secondary staining with anti-human IgG1-PE (R&D systems). Control cells were prepared in the same fashion except that hIgG1 Fc (R&D systems) was used instead of mCD47-Fc. Flow cytometry was performed to measure mCD47-Fc binding to monocyte subset (CD45⁺Lin⁻Ly6G⁻CD11b⁺CD11c⁻F480⁻ cells). SIRPα expression was performed by staining cells with PE-conjugated P84 antibody (BD) under identical conditions as binding assay.