Anti cd32 fitc

To assess surface FcgRIIa expression, 2e5 cells per cell type and stain were washed in FACS wash (FW, 2% FBS in 1x PBS) and resuspended in 50 μL anti-CD32-FITC (Cat # 60012.FI, StemCell) or isotype control (Cat # 11–4732-81, ThermoFisher Scientific), and incubated at 4°C for 20 min. FW was then added and a wash with FW was performed. Cells were then fixed in 2% PFA for 20 minutes, spun down, and resuspended in PBS at 4°C until acquisition. To assess total FcgRIIa expression, 2e5 per cell type and stain cells were fixed using Cytofix (Cat #554655, BD) for 20 minutes at 4°C cells before addition of perm/wash buffer (Cat #554723, BD) followed by two washes in perm/wash. Cells were then resuspended in either 50 μL anti-CD32-FITC or isotype control, and incubated at 4°C for 20 min. Cells were then washed with perm/wash prior to resuspension in PBS at 4°C until acquisition. Samples were analyzed via flow cytometry (Sony ID7000), and data were analyzed using FlowJo 10.9.0.

K562 cells and U937 cells were washed in FACS wash (FW, 2% FBS in PBS) and resuspended in 50 μL of staining or isotype control antibody and incubated at 4°C for 30 min. For FcγRII we stained with anti-CD32-FITC (Cat# 60012.FI, StemCell) and corresponding mouse IgG2b-FITC isotype control (Cat# 11-4732-81, ThermoFisher Scientific). For FcαRI we stained with anti-CD89/-PE (cat# 555686, BD Biosciences) and corresponding mouse IgG1-PE isotype control (cat# 12-4714-42, ThermoFisher Scientific). Cells were washed twice in FW and analyzed by flow cytometry.