Primary mouse hepatocytes were isolated from 9- to 12-week-old mice, as described previously (95). Briefly, the liver was perfused with Earle’s balanced salt solution (Invitrogen) containing 0.5 mM EGTA for 5 min and perfused with type I collagenase (200 U/ml; Worthington) via the inferior vena cava for 5 min. After dissection, hepatocytes were released by scattering, passed through a 100-μm cell strainer, and then spun at 50g for 1 min. The pellet was resuspended in DMEM and then spun at 50g for 10 min in a Percoll gradient to remove dead hepatocytes. Viable cells were washed with DMEM at 50g for 10 min and checked by trypan blue staining. Primary mouse hepatocytes were plated in DMEM with 10% FBS.

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