Novocyte 3001

For surface marker staining, cells were blocked with Fc Shield (anti-mouse CD16/CD32, Tonbo) for 10min then incubated with antibodies of interest for 30 min. Samples were washed extensively with sterile FACS buffer (PBS, 2% FCS, 2mM EDTA) then analyzed using Novocyte 3001 (ACEA Biosciences). Cells were gated on singlets and dead cells were excluded using Zombie Yellow live/dead staining (Biolegend). Data were analyzed using FlowJo software (BD).
For cell sorting, samples were stained, washed, then sorted by Moflo XDP (Beckman Coulter) directly into 10% FCS containing complete RPMI media. Cells were washed once with complete RPMI media before culturing.

Antibodies used for flow cytometry are listed in Table S2. For surface marker staining, cells were blocked with Fc block (anti-mouse CD16/CD32, BioLegend) for 10 min and then incubated with antibodies for 30 min, with extensive washes with FACS buffer (PBS, 2% FCS, and 2 mM EDTA). For intracellular cytokine analysis, cells were stimulated with 50 ng/ml PMA (Sigma-Aldrich) and 1 µM ionomycin (Sigma-Aldrich) in the presence of 5 µg/ml brefeldin A (BioLegend) for 4–6 h. Cell permeabilization and intracellular staining were performed according to manufacturer’s protocol (eBioscience Foxp3/TF staining buffer set). Samples were analyzed using an LSR Fortessa flow cytometer (BD) or Novocyte 3001 (ACEA Biosciences). Cells were gated on singlets, and dead cells were excluded using Zombie Yellow live/dead staining (BioLegend). Data were analyzed using FlowJo software (BD). For FACS sorting, cells were stained in sterile FACS buffer. Dead cells were excluded by DAPI or propidium iodide staining. Cells were sorted by FACS Aria (BD) into FACS buffer or directly into TriZol LS Reagent (Invitrogen) for RNA extraction.