Primary murine hepatocytes obtained from WT mice were isolated24,25,31 and cultured and maintained in regular DMEM growth media (Sigma, #D5796) containing 10% FBS. For in vitro starvation experiments, “starved” media contained 1 g/L glucose and 0.5% FBS was used. Cultures were lysed in Trizol and subjected to downstream analysis. In vitro genetic knockdown was achieved via siRNA transfection using Lipofectamine 3000 from Invitrogen (L3000015). Trehalose was obtained from Sigma Aldrich (St. Louis, MO) and was >97% purity by HPLC. Three percent trehalose water (w/v) fed ad libitum was used in all in vivo experiments.
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.