Cell dispersion solution

Islets and pLNs were harvested from female NOD mice of various ages. Handpicked islets were dispersed using a non-enzymatic cell dispersion solution (Sigma) for single-cell suspensions. Total cells from the pLNs were isolated by digestion with Liberase (125 ug/ml, Roche) and DNase (50 ug/ml, Roche). Cells from islet and pLN cells were mixed together and cultured in DMEM media supplemented with 10% fetal bovine sera at a concentration of 2×10⁶ cells/ml. The culture was supplemented with 20 U/ml IL-2 and 1 uM of the peptide of interest (challenge). After incubation at 37°C for 7 days, total live cells were collected from the culture using Histopaque 1119 and were subject to a second round of amplification. Accordingly, live cells were added to a fresh culture containing 20 U/ml IL-2 and 1 μM peptides as before along with irradiated NOD splenocytes (3000 RAD) as APCs (2 × 10⁶/ml). After incubation at 37°C for 3 days, cells were harvested and were assayed for reactivity by eliciting a recall response on either IL-2 or IFN-γ coated 96-well multi-screen plates (MilliporeSigma) for ELISPOT assay. Cultured cells were plated as 2.5 × 10⁵ /well with 10 μM antigen (recall), and the reactive cells were counted using Immunospot Software.