Cd16 32 fc blocking antibody

Single-cell suspensions were blocked with CD16/32 Fc blocking antibody (Cat# 101320, Biolegend) for 15 min and incubated with antibodies against surface markers for 30 min at 4°C, followed by 3 washes in FACS buffer. These samples were then run on an LSR II Green flow cytometer (BD Biosciences) and analyzed by FlowJo software (BD Biosciences).
Intracellular transcription factor staining was performed after surface staining. The cells were fixed with buffer from the True-Nuclear Transcription Factor Buffer Set (Cat# 424401, BioLegend) for one hour according to the manufacturer’s protocol. Cells were then washed with permeabilization buffer and stained with antibodies against transcription factors in permeabilization buffer.
For intracellular cytokine staining, single-cell suspensions were stimulated with anti-CD3/CD28 (Cat# 100340 and 102116, respectively, Biolegend) in the presence of brefeldin A (Cat# 420601, BioLegend) and 10 ng/mL IL
2 (Cat# 212–12, Peprotech) for 6 hours at 37°C. Subsequently, cells were collected for staining of surface markers and fixed by fixation buffer. Cells were then washed with permeabilization buffer and stained for cytokine-specific antibodies in permeabilization buffer.
All antibodies used for flow cytometry are listed in Supplemental Table 1.