B220 efluor450 ra3 6b2

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.

Single-cell suspensions of splenocytes were treated with ACK lysis buffer to remove RBCs before staining in PBS, containing LIVE/DEAD fixable near-IR dead cell stain (Life Technologies) and appropriate, pretitered Abs. Abs used, indicating Ag and fluorophore (and clone): CD69-FITC (H1.2F3), CD86-PE (GL-1), streptavidin-PerCP (Becton Dickinson), MHC class II-bio (I-Ab; M5/114.15.2), IgM-PECy7 (II/41), B220-eFluor450 (RA3-6B2; eBioscience), CD19-FITC, B220-PerC, IgM-PE, IgG1-APC, PNA-FITC, CD38-PE/Cy7, CD45.2-ef450, CD45.1-FITC, CD138-PE, IgD-ef450, CD4-FITC, CD8-FITC, CD45.2-PerCP-cy5.5, CD45.1-FITC, B220-BV605, CD4-BV650, CD8a-BV650, CD38-bio, Streptavidin-PacO, FAS-PE. Anti-Syk Ab (5F5; BioLegend) and NIP-BSA were labeled using LYNX Rapid APC, RPE, and PerCP Ab Conjugation Kits (AbD Serotec). Ab staining of intracellular Syk was performed as described previously (13 (link)).