Pancreatic Islet and Spleen Single-Cell Isolation

Pancreatic islets were isolated as previously described (Foda et al, 2020 (link)). Briefly, pancreatic islets were isolated by perfusing the pancreas via the common bile duct with collagenase P solution (0.5 units/ml). The inflated pancreata were incubated for 16 min at 37°C, agitated, and washed with Hank’s balanced salt solution (Sigma-Aldrich) plus 2% fetal bovine serum. Islets were hand-picked from the pancreas digestion slurry and dissociated in non-enzymatic cell dissociation buffer. Islet cell suspensions were resuspended in RPMI and incubated at 37°C for 1 h. Final islet single cell suspensions were washed and resuspended in RPMI. Spleens were harvested and mechanically processed with a 70 μm filter to obtain a single-cell suspension. Red blood cell lysis was performed using ACK lysing buffer (Lonza). Splenocytes were enriched for CD8 T cells using magnetic bead-based negative selection (CD8 mouse T-cell isolation kit; Miltenyi Biotec). Final spleen single-cell suspensions were washed and resuspended in RPMI. Islets and spleens were each pooled from 10 mice for both scRNA-seq experiments.