Dissected liver tissues were incubated for 30 min at 37°C in dissociation medium composed of Leibovitz-15 medium (Sigma-Aldrich), 45% glucose, 1 g/L DNAse I (Applichem) and collagenase A (Roche, Basel, Switzerland). Digested liver fragments were passed through a nylon cell strainer, and cells free in suspension were collected in 50 ml wash medium/liver and stored at 4°C. Cells were centrifuged at 30 × g for 15 min at 4°C to pellet parenchymal cells, i.e., hepatocytes. Parenchymal cells were washed 3 times with FACS buffer and analyzed by flow cytometry.
Non-parenchymal cells (NPCs) remaining in suspension were collected and centrifuged at 300 × g for 10 min at 4°C. Pelleted cells were resuspended in ice-cold-HBSS, mixed with freshly prepared 30% Histodenz (Sigma-Aldrich), and centrifuged at 1,500 × g for 20 min at 4°C. The NPCs at the Histodenz interface were collected, washed, and suspended in FACS buffer for analysis. The NPCs were phenotyped using dye-conjugated monoclonal antibodies specific for CD45 (clone 30-F11), F4/80 (clone BM8), and CD31 (clone 390) (BD Biosciences, Franklin Lake, NJ, United States). Stained samples were acquired on a multichannel cytometer BD LSR II equipped with FACS Diva software (BD Biosciences; Franklin Lake, NJ, United States) and analyzed using FlowJo 7.6.5 (Becton, Dickinson Company; Ashland, OR, United States).
Non-parenchymal cells (NPCs) remaining in suspension were collected and centrifuged at 300 × g for 10 min at 4°C. Pelleted cells were resuspended in ice-cold-HBSS, mixed with freshly prepared 30% Histodenz (Sigma-Aldrich), and centrifuged at 1,500 × g for 20 min at 4°C. The NPCs at the Histodenz interface were collected, washed, and suspended in FACS buffer for analysis. The NPCs were phenotyped using dye-conjugated monoclonal antibodies specific for CD45 (clone 30-F11), F4/80 (clone BM8), and CD31 (clone 390) (BD Biosciences, Franklin Lake, NJ, United States). Stained samples were acquired on a multichannel cytometer BD LSR II equipped with FACS Diva software (BD Biosciences; Franklin Lake, NJ, United States) and analyzed using FlowJo 7.6.5 (Becton, Dickinson Company; Ashland, OR, United States).