Cd45 percpcy5.5 30 f11

The detection of B cells with receptors that bind insulin has been previously described (31 (link)). Briefly, single-cell suspensions isolated from the thymus were incubated overnight at 4°C in PBS supplemented with 1% fetal bovine serum, 1% anti-CD16/32 antibodies (eBiosciences) and biotinylated insulin (0.1 μg/10⁶ cells, ibt systems). Bound insulin was detected with fluorochrome-labeled streptavidin Alexa 6470 (Invitrogen) for 30 min at 4°C. The cells were subsequently incubated with anti-CD19 PE (6D5; eBiosciences), B220 eFluor450 (RA3-6B2; eBiosciences), -CD4 BV650 (GK1.5; BioLegend), CD8β PE-Cy7 (H35-17.2; eBiosciences), -CD45 PerCPCy5.5 (30-F11; eBiosciences) antibodies and LIVE/DEAD Fixable Dead Cell Stains (Thermo Fisher Scientific) for 30 min at 4°C, following which the cells were analyzed by flow cytometry. B cell gates were defined following exclusion of dead cells and T cells (dump channel). All samples were stained with insulin-biotin followed by streptavidin or with streptavidin only, frequencies of B cells insulin⁺ were calculated subtracting the background calculated in sample-matched streptavidin only control.

For single-cell sequencing experiments, single-cell suspensions were stained for 30 min at room temperature with the following antibodies: O4-APC (O4, 10 µL/test, Miltenyi, 130-095-891), CD11b-e450 (M1/70, 0.5 µL/test, eBioscience, 48-0112-82), TER119-APC/Cy7 (TER-119, 1.25 µL/test, Biolegend, 116223), PDGFRα-PE/Cy7 (APA5, 0.625 µL/test, Invitrogen, 25-1401-82), CD45-PerCP/Cy5.5 (30-F11, 0.5 µL/test, eBioscience, 45-0451-82), and CD16/31 (93, 0.5 µL/test, Invitrogen, 14-0161-82). Viability was determined using Ghost Dye Violet 510 (0.5 µL/test, Tonbo biosciences, 13-0870). Cells were sorted using a 16-color BD influx cell sorter. Cells used for sequencing were gated on live/singlets/TER119−/CD45−/CD11b−/YFP+. Following sorting, cells were washed three times with 0.04% BSA and then processed for sequencing according to the 10× Genomics protocol.