Collagenase 5

Human PaC cell lines SW1990 and BxPC-3 were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China), and the mouse PaC cell line PAN02 was purchased from the Shanghai Fu Heng Biology (Shanghai, China). All the cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS, Gibco) and were grown in a humidified 5% CO₂ atmosphere with a temperature of 37 °C.
Mouse pancreatic β cell line MIN6 (obtained from Jiangsu Engineering Research Center for MicroRNA Biology) cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 15% FBS and 100 µmol/L 2-mercaptoethanol (Sigma, M3148). Primary mouse islets were isolated from wild-type male 6~8 week-old C57BL/6J mice by collagenase V (Sigma) digestion and Histopaque (Sigma) as described previously 17 (link), hand-picked, and cultured, prior to further investigations, for at least 12 h in RPMI 1640 medium containing 10 mmol/L glucose, 15% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Both MIN6 cells and primary pancreatic islets were grown in a humidified 5% CO₂ atmosphere with a temperature of 37 °C.

Four weeks post-vaccination or eight weeks post-infection, lungs were aseptically removed, cut into small pieces, and then digested in 3 mL digestion media (RPMI 1640 containing 50 μg/mL DNase I (Sigma, USA), 1 mg/mL Collagenase V (Sigma, USA), 5% fetal bovine serum, 100 U/mL Penicillin and 100 μg/mL Streptomycin (Solarbio, China) for 1 h at 37°C with 5% CO₂. Suspensions were passed through a 70 μm cell strainer to obtain a single cell suspension. Cells were pelleted by centrifugation and lysed with red blood cell lysis buffer. After being washed with RPMI 1640 medium, cells were resuspended and adjusted to a suitable density in complete RPMI 1640 medium.

Single cell suspensions of small and large intestines (colon) from non-perfused mice were prepared for flow cytometry. Intestines were opened longitudinally and cut into 2–3 cm segments in ice-cold PBS and intestinal contents removed by gentle shaking. Tissue segments were incubated at 37 °C for 20 min whilst shaking in RPMI containing 3% endotoxin-free FBS (GE Healthcare), 20 mM HEPES (Gibco), 5 mM EDTA (Sigma), 1 mM DTT (Promega), and 100 U/mL polymyxin B (Sigma). Segments were transferred to RPMI containing 2 mM EDTA and 20 mM HEPES and shaken by hand to ensure optimal dissociation of epithelial cells and lamina propria leukocytes. Tissues were minced with scissors and digested at 37 °C for 30 min, whilst shaking in RPMI containing 20 mM HEPES, 0.425 mg/mL Collagenase V (Sigma), 0.625 mg/mL Collagenase D (Roche), 1 mg/mL Dispase (Gibco), and 30 µg/mL DNase (Roche). Cell suspensions were passed through 70 µm then 40 µm cell strainers in RPMI containing 10% endotoxin-free FBS and 100 U/mL polymyxin B. After centrifugation at 400×g at 4 °C for 5 min, including a wash step, cells were resuspended in PBS containing 2% FBS for flow cytometry.