Detection of Insulin-Binding B Cells

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.