Isolation of Murine Immune Cell Subsets

Pancreata and spleens were harvested at indicated time points and mechanically disrupted in RPMI. Pancreata were then subjected to enzymatic digestion using RPMI + 10%FBS + 3.6 mg/mL collagenase D (Roche, Basel, Switzerland). Red blood cells from both pancreata and spleens were lysed using red blood cell lysing solution (eBioscience) and the resultant suspension was filtered using a 70-µm cell strainer to remove any resultant debris. Cells were then stained for intracellular and surface antigens as previously described (Surana et al 2014 (link)). The following antibodies were used for surface antigen staining:CD45 (Biolegend; clone 30-F11), CD4 (eBioscience; clone RM4-5), CD8a (Biolegend; clone 53-6.7), CD3 (Biolegend; clone 145-2C11), F4/80 (Biolegend; clone BM8), MHCII (Biolegend; cloneM5/114.15.2), Gr1(Biolegend; clone RB6-8C5), CD11b (Biolegend; clone M1/70), CD11c (Biolegend; clone N418), CD19 (BD Bioscience; clone1D3). In order to isolate CD45⁺CD3⁺CD8⁺ CTLs and CD45⁺CD3⁻F4/80⁺ macrophages, CD45⁺ leukocytes were first divided into CD3⁺ or CD3⁻ fractions, and the CD8⁺CD4⁻ subpopulation among CD45⁺CD3⁺ cells and the F4/80⁺ subpopulation within CD45⁺CD3⁻ cells were collected using BD FACSAria II (BD Biosciences).