Flow Cytometry Staining and Analysis

Cells were stained for flow cytometry and included a pre-incubation step with unconjugated anti-CD16/32 (clone 93) to block Fc receptors as previously described (47 (link), 48 (link)). The antibody cocktails used for different sets of stains are listed in Supplementary Table 1. For viability staining, DAPI (Sigma-Aldrich, 0.005 μg/ml final concentration) or propidium iodide (Sigma-Aldrich, 0.025 μg/ml final concentration) was used. Single color stains were used for setting compensations and gates were determined with fluorescent-minus one controls, isotype-matched antibody controls, or historical controls. Intracellular staining of Ki67 was performed using the eBioscience™ Foxp3/Transcription Factor Staining Buffer Set following the manufacturer’s instructions. For cell cycle analysis, DAPI was used at a final concentration of 0.1 μg/ml per sample. Apoptosis staining was performed using Biolegend Annexin V Apoptosis Detection Kit with 7AAD. Flow cytometry data was acquired on the BD LSR II. The data was analyzed using FlowJo Software version 10.7.1.