Liver digest buffer

The reagents for primary hepatocyte isolation and culture including Medium 199, Dulbecco’s Modification of Eagle Medium (DMEM), liver perfusion buffer and liver digest buffer were obtained from Invitrogen (Carlsbad, CA). For RNA extraction, RNA STAT-60 was purchased from TEL-TEST (Friendswood, TX). The reagents for cDNA synthesis and real-time PCR were obtained from Applied Biosystems (Foster city, CA). All real-time PCR primer sets used in this study were synthesized by Sigma-Aldrich (St. Louis, MO). Antibodies against β-actin (#4967), PKCζ (#9368), PKCι (#2998), phospho- PKCζ/λ (p-PKCζ/λ) Thr410/403 (#9378), AKT (#9272), phospho-AKT (p-AKT) Ser473 (#9271), p-AKT Thr308 (#9275), p-AKT Thr450 (#9267), insulin receptor substrate 1 (IRS1, #2382), fatty acid synthase (FAS, #3189), acetyl CoA carboxylase (ACC, #3662), phospho-ACC (p-ACC) Ser79 (#3661) used in this study were purchased from Cell Signaling Technology (Danvers, MA). All other reagents and materials were purchased from Fisher Scientific (Pittsburgh, PA) unless described otherwise.

Hepatocytes were isolated from mice using a modified two-step perfusion method using Liver Perfusion Media and Liver Digest Buffer (Invitrogen). Cells were seeded on plates (pre-coated (1 h) with collagen I (BD Biosciences)) in DMEM plus 10% FBS, 2 mM sodiumpyruvate, 1 μM dexamethasone and 100 nM insulin plus 2% penicillin/streptomycin. After attachment (3 h), the medium was removed and the hepatocytes were incubated (22 h) in maintenance medium (DMEM (4.5 g l⁻¹ glucose) supplemented with 10% FBS, 0.2% BSA, 2 mM sodium pyruvate, 2% Pen/Strep, 0.1 μM dexamethasone, 1 nM insulin) before stimulated with insulin at 100 nM insulin for 30 min.

The reagents for primary hepatocyte isolation and culture including Medium 199, Dulbecco's Modification of Eagle Medium (DMEM), liver perfusion buffer and liver digest buffer were obtained from Invitrogen (Carlsbad, CA). For RNA extraction, RNA STAT-60 was purchased from TEL-TEST (Friendswood, TX). The reagents for cDNA synthesis and real-time PCR were obtained from Applied Biosystems (Foster city, CA). All real-time PCR primer sets used in this study were synthesized by Sigma-Aldrich (St. Louis, MO). All other reagents and materials were purchased from Fisher Scientific (Pittsburgh, PA) unless described otherwise.