Isolation and Manipulation of Sirt1/3-Deficient Hepatocytes

Primary hepatocytes from Sirt1 floxed (Sirt1^L2/L2) or Sirt3 floxed (Sirt3^L2/L2) mice (described in (21 (link)) and (23 (link)), respectively) were isolated with Liberase Blendzyme (Roche) perfusion as described previously with minor modifications (24 ). Isolated hepatocytes were then plated in DMEM (Gibco; 4.5 g/l glucose) with 10% FBS and penicillin and streptomycin and maintained at 37°C in a 5% CO₂ atmosphere. Four hours after plating, cells were transduced with Ad-GFP or an Ad-Cre virus at a MOI = 5 to generate matched Sirt1^−/− or Sirt3^−/− (loss-of-function) and Sirt1^L2/L2 or Sirt3^L2/L2 wild-type hepatocytes, respectively. Hepatocytes were then incubated overnight and before changing media. Primary hepatocytes were then treated with 1mM NR or vehicle (ddH₂0) for 24 hrs.