Isolation and Culture of Primary Hepatocytes

Primary hepatocytes were cultured according to the published methods,^{43 (link)} with a slight modification. Liver perfusion medium (Invitrogen, 17701-038) was used to perfuse the livers in situ via the portal vein and was followed by liver digest medium (Invitrogen, 17703-034) after anesthetizing the mice with pentobarbital sodium. The liver was excised and minced in William's E medium (Life technologies, A12176-01, Grand Island, NY, USA). The cell suspension was mixed gently several times with a pipette and strained through a steel mesh sieve after removing the liver capsule. The dispersed hepatocytes were collected via centrifugation at 50 × g for 5 min at 4 °C and washed twice with William's E medium. Hepatocytes were isolated by Percoll separation and washed twice with William's E medium. The final pellet was resuspended in William's E medium. The hepatocytes were counted, and their viability was determined by trypan blue exclusion. The hepatocytes were cultured under normoxic conditions (air/5% CO₂) for further experiments.