Ketamine xylazine mixture

Age-matched C57BL/6J albino males between 3 and 4 months were used for PH culture isolation. All mice were anesthetized using a ketamine/xylazine mixture (Henry Schein) before isolation. The hepatic portal vein was cannulated and perfused with Kreb’s Ringer Bicarbonate Buffer (Sigma-Aldrich) containing EGTA for 10 min at 37 °C. After the first wash, a second Kreb’s Ringer wash containing CaCl₂ and Liberase (Roche Holding AG) was applied for 10 min at 37 °C. Hepatocytes were filtered through a gauze mesh and resuspended in plating media: William’s Media E (Sigma-Aldrich) containing 10% fetal bovine serum (Sigma-Aldrich), 200 nM dexamethasone (Sigma-Aldrich), 1 × penicillin/streptomycin (Thermo Fisher Scientific), and 2 mM L-glutamine (Thermo Fisher Scientific). Cells were plated at a density of 3 × 10⁵/ml on a six-well collagen (Sigma-Aldrich)-coated plate. Hepatocytes were allowed to recover overnight and all experiments were started 24 h post isolation, unless otherwise indicated.

Age-matched C57/B6J albino females between 3 and 4 months were used for primary culture isolation. Mice were first anesthetized using a ketamine/xylazine mixture (Henry Schein Inc.). To obtain primary hepatocytes, the hepatic portal vein was cannulated and perfused with Krebs-Ringer solution containing EGTA for 10 min at 37 °C. After the first wash, a second Krebs-Ringer wash containing CaCl and Liberase^TM (Roche) was used for 10 min at 37 °C. Hepatocytes were filtered through a gauze mesh and resuspended in plating media: Williams' Media E (Sigma);10% FBS (Sigma); 200 nm dexamethasone (Sigma); 5 ml penicillin/streptomycin (Fisher); and 2 mml-glutamine (Fisher). Cells were plated at a density of 0.3 × 10⁶ on 6-well collagen-coated (Sigma) plate. Hepatocytes were allowed to recover overnight and experiments were started 24 h post isolation.