Biotinylated human insulin

Single cell suspensions from spleen and PLNs were prepared using HBSS (Invitrogen Life Technologies) + 10% FBS (HyClone). Spleen RBCs were lysed using Tris-NH₄Cl. Cells were subsequently stained for flow cytometry analysis in 1X PBS containing 0.1% sodium azide, 0.02% EDTA, and 1% FBS using the following reagents and reactive antibodies (BD Biosciences or eBiosciences): Fixable Viability Stain 700 for cell viability, B220 (6B2), CD19 (1D3), CD21 (7G6), CD23 (B3B4), IgM (15F9), IgD (11–26c), IgM^a (DS-1), IL-21 (mhalx21) and IL-10 (JES5–16E3, BioLegend). Insulin-binding BCRs were detected using biotinylated human insulin (Sigma-Aldrich) (26 (link)). Insulin-occupied BCRs were detected using biotinylated anti-insulin mAb123 (ATCC #HB-123) (27 (link)). The insulin epitope recognized by mAb123 is distinct from the mAb125 insulin epitope used to derive V_H125.Tg. mAb123 detects anti-insulin V_H125^SD.NOD BCR occupancy with endogenous insulin, but not insulin bound to the insulin receptor (25 (link), 28 (link)). Avidin-fluorochrome conjugates (BD Biosciences) were used to detect biotinylated reagents. Samples were acquired using a BD Biosciences LSR II flow cytometer, and FlowJo software (Tree Star, Inc) was used for data analysis.

Cells were isolated from spleens and pancreata as previously described (36 (link)). Lymph nodes were macerated in HBSS (Invitrogen Life Technologies) + 10% FBS (HyClone). Cells were subsequently stained for flow cytometry analysis using the following reagents and reactive antibodies: 7-aminoactinomycin D (7-AAD), B220 (6B2), BCL-6 (K112–91), CCR6 (140706), CD11b (M1/70), CD11c (HL3), CD4 (RM4–5), CD44 (IM7), CD69 (H1.2F3), CD95/FAS (JO2), CD278/ICOS (C398.4A), CXCR3 (CXCR3–173), CXCR5 (2G8), eFluor Fixable Viability Dye, GL7, IgD (11–26c), IgM^a (DS-1), IgM^b (AF6–78), IgM (μ chain specific), NKG2D (cx5), or PD-1 (J43) (BD Biosciences, eBioscience, ThermoFisher Scientific, or Tonbo Biosciences). Biotinylated human insulin (Sigma-Aldrich) (33 (link)) was used to detect insulin-binding specificity. Avidin-fluorochrome conjugates were used to detect biotinylated reagents. Samples were acquired using a BD Biosciences LSR II flow cytometer and data were analyzed using FlowJo software (Tree Star, Inc).