Apc labeled anti ifn γ xmg1.2

Infiltrating lymphocytes in islets were isolated as described [24 (link)]. Briefly, pancreatic tissues were collected and digested with Liberase RI (Roche Applied Science) at 37°C for 15–20 min. Dissociated islets were isolated under a dissecting microscope. To release intra-islet lymphocytes, islets were digested with Trypsin-EDTA for 10 min, followed by treatment with cell dissociation buffer (GIBCO/BRL) for 15 min at 37°C. After overnight incubation at 37°C, 4-5x10⁴ cells (70–80% viable) were recovered from pooled pancreata from 5 mice. Lymphocytes were then fixed and externalization of CD107a (1D4B, Biolegend) was determined by flow cytometry. To detect IFN-γ production, lymphocytes were re-stimulated with 1μM OVA peptide (SIINFEKL) in the presence of Golgi blocker (BDbiosciences) for 5 hours. Cells were fixed and permeabilized according to the manufacturer's instructions. The level of intracellular IFN-γ was detected by APC labeled anti-IFN-γ (XMG1.2, Biolegend). To detect T-bet and pStat5, cells were fixed using BD Phosflow Lyse/Fix Buffer and stained with PE labeled anti-T-bet (4B10, eBioscience) and anti-pSTAT5 (pY649, BDbiosciences), respectively.