Isolation of Hepatic Mononuclear Cells

Hepatic mononuclear populations were isolated as previously described (Montes de Oca et al., 2016a (link); Stanley et al., 2008 (link)). Briefly, mice were sacrificed by CO₂ asphyxiation after which the liver was perfused by injecting 5-10 mL of 1x PBS through the portal vein. The liver was excised, weight and collected into 10 mL 2%FBS.PBS. Livers were passed through a 200 μm metal mesh held inside a tea strainer using the back of a 5 cc/ml syringe plunger (Terumo Medical). Mesh was washed with 10-15 mL 2%FBS.PBS and cell suspension was made up to 40 mL and centrifuged at 350 g in an Eppendorf Centrifuge 5810 R (Thermo Fisher Scientific). Hepatocytes were separated form leucocytes using a 33% (v/v) Percoll Density Gradient Media (GE Healthcare, Little Chalfond, UK) and centrifuged at 575 g for 15 minutes at RT with the brake off. The leucocyte cell pellet was incubated in 1 mL Red Blood Cell Lysis Buffer Hybri-Max (Sigma-Aldrich) for 6 minutes at RT. Cells were washed again in 10 mL 2%FBS.PBS after which cell pellet was diluted in DPBS (GIBCO) and Trypan Blue Stain (Invitrogen) and counted using Countess Cell Counting Chamber Slides on the Countess II FL (both from Invitrogen), as per manufacturer’s protocol.