Trypsin inhibitor

KCs were prepared by collagenase liver perfusion and differential density centrifugation. Briefly, the liver was perfused in situ with a HEPES based buffer containing 0.015% collagenase (Sigma) and 0.004% trypsin inhibitor (Wako Pure Chemical Industries Ltd., Osaka, Japan). After excision, the hepatic cells were suspended in Hanks' balanced salt solution and filtered through a nylon mesh. The filtrate was centrifuged thrice at 50 ×g (4°C) for 90 s to delete the parenchymal hepatocytes. Flow cytometric analysis involved centrifugation of purified fractions of nonparenchymal cells (NPC) from the liver, on a layer of 20% and 50% Percoll, at 1,000 ×g (4°C) for 20 min in order to remove dead cells. The suspension containing KCs was collected from the intermediate layer and resuspended in PBS containing 5 mM EDTA and 1% FBS, for antibody binding as described [7 (link)].

For the KC phagocytosis assay, we isolated non-parenchymal cells from livers one day following exercise. The mice were anesthetized and perfused with liver perfusion medium (Gibco) for 4 min, followed by DMEM with collagenase Type 4 (Worthington Biochemical), trypsin inhibitor (Wako Chemical), and HEPES (Dojindo) at 37 °C for 10 min. Liver samples were collected and cells were isolated in hepatocyte wash medium (Gibco). To remove parenchymal cells, the samples were centrifuged three times at 30 g for 2 min. The supernatants were centrifuged at 400 g for 8 min, and precipitates including KCs were obtained. The samples were analysed by flow cytometry.

Mice pancreatic acinar cell culture was performed as previously described³⁹ . Briefly, the pancreas was removed and minced in small pieces. Then the pancreatic segments were enzymatically digested in 10 mL HBSS (Nacalai Tesque, #17461-05) with 10 mM HEPES (Gibco, #15630-080), 200 U/mL collagenase (Wako), and 0.25 mg/mL trypsin inhibitor (Wako, #202-09221) for 30 min at 37 °C. During incubation, mechanical digestion using serological pipettes was performed every 10 min. After washing, pancreatic acini were resuspended in 7 mL of Waymouth’s medium (Gibco, #11220-035) containing 2.5% fetal bovine serum (HyClone), 10 mM HEPES, 0.25 mg/mL trypsin inhibitor and 25 ng/mL recombinant human epidermal growth factor (PEPROTECH, #315-09), seeded on type I collagen coated six-well plate and cultured at 37 °C overnight.
Cultured pancreatic acini from control of Ptf1a^Cre-ERTM-tdTomato mice was supplemented with 10 μM of 4-hydroxy Tamoxifen (4OHT) (Sigma-Aldrich, #H7904).
For stimulation experiments, 200 ng/mL LPS derived from E. coli (Sigma-Aldrich, #L2630), 20 ng/mL TNF (BioLegend, #575204), 20 ng/mL IL-6 (BioLegend, #575702), or 100 ng/mL RANKL (Wako, #184-01791) were added in medium representatively and cultured at 37 °C overnight. The stimulated pancreatic acini were resuspended in TRIzol and performed qPCR described above.