Flow Cytometry Surface Staining Protocol

Flow cytometry experiments were performed as previously described (36 (link), 50 (link)). For surface staining, up to 0.5x10⁶ cells were incubated with 10% FBS in 1X PBS for 10 minutes. Cells were then stained with 100 μl of PBS containing 0.1 μl fixable viability dye eFluor506 (eBioscience, corresponding to 1:1000 dilution of the stock, as per the manufacturer’s recommendation), 2 μl of FcR blocking reagent (Biolegend, corresponding to 1:50 dilution of the stock; we validated internally that this dilution is performing equally to the manufacturer’s recommended dilution of 1:20), and various combinations of the antibodies listed in Table S2 for 20 min at room temperature. For single-cell RNA sequencing, TotalSeq™-C oligonucleotide-conjugated antibodies (Biolegend) were also added at this step at 0.01mg/mL final concentration. After two washes in PBS, cells were resuspended into 100 μl of MACS buffer (PBS containing 2mM EDTA (pH 8.0) and 0.5% BSA) and stored at 4°C protected from light for up to 4 hours until flow cytometry acquisition.