Ham s f 12 nutrient mixture

PSCs were isolated from the pancreas of male Wistar rats weighing 200–250 g by density–gradient centrifugation.^{30 (link)} Cell purity was assessed by a typical star-like configuration and by detecting vitamin A autofluorescence and always exceeded 95%. Isolated stellate cells were resuspended in complete Dulbecco’s modified Eagle medium mixed with Ham’s F-12 nutrient mixture (Wako Pure Chemicals, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum, 50 units/mL penicillin, and 50 mg/mL streptomycin (Invitrogen). Upon reaching confluence, the cells were trypsinized with 0.05% trypsin/0.01% EDTA (Invitrogen) and passaged at a ratio of 1:3. All experiments were performed using third-passage PSCs, except for those using freshly isolated PSCs (first passage; 2 days after isolation). Unless specified otherwise, PSCs were incubated in serum-free medium for 24 hours before the addition of experimental reagents. PA was dissolved in medium, making an emulsion with BSA at a molar ratio of 10:1. Fresh PA/BSA complex was prepared before each experiment. We examined the effects of BSA as a control to clarify whether the effects of PA were due to nonspecific protein binding to PSCs. For some experiments, GSK2606414, a specific inhibitor of PERK phosphorylation,^{31 (link)} was added 1 hour before PA or BSA treatment.

PSCs were isolated from the pancreas of normal male Wistar rats (weighing 180–200 g) by a density-gradient centrifugation method as previously described ^{56 (link)}. Briefly, purity of PSCs was more than 90% as evaluated by the typical star-like configuration and by detecting vitamin A autofluorescence. Cells were maintained in complete DMEM/F-12: this is a mixture of DMEM (Dulbecco’s modified Eagle medium) and Ham’s F-12 nutrient mixture (from Wako Pure Chemicals, Osaka, Japan) supplemented with 10% fetal bovine serum, 50 units/mL of penicillin, and 50 mg/mL of streptomycin (from Invitrogen, Carlsbad, CA, USA). All experiments using PSCs were performed with cells between passages 1 and 5. Unless otherwise specified, PSCs were incubated in serum-free medium for 24 h before the addition of experimental reagents.