Pancreas Single-Cell Isolation and Sorting

For mouse pancreas dissociation into single cell suspensions, pancreata were minced with scissors and incubated with trypsin-EDTA for 10 min at 37°C, followed by treatment with collagenase P (Sigma, 11213857001) 1 mg/mL for 20–30 min at 37°C, and then filtered through 70 and 40 μm filters. Purified cells were incubated with the fluorescent β-galactosidase substrate C₁₂FDG (Invitrogen D-2893) at 33 µM for 1 hour at 37°C, followed by sorting based on C₁₂FDG and tdTomato or Epcam fluorescence, using an Aria III sorter (BD Biosciences), with gating for CD31^– and CD45^– cells to exclude endothelial and immune cells. Imaging flow cytometry was performed as previously described,42 (link) and imaged by ImageStreamX flow cytometry (Amnis). Antibodies used for FACS: Cd45 (eBiosciences 25-0451-82), Cd31 (eBiosciences 17-0311-80), Epcam (Biolegend 118212), Cd140a (Biolegend 135919), Pdpn (Biolegend 127423), F4/80 (Biolegend 123117) and CK19 (DSHB TROMA III).