Dowex 1x2 400 ion exchange resin

MEFs (10⁵/well) were plated in six-well plates, allowed to adhere overnight, and then pre-incubated in either serum-containing or serum-free medium for 2 hours. Cells were then changed to medium containing 1μCi [9,10(n)-³H]palmitic acid (Perkin-Elmer, Cat# NET043001MC) or 1.8 μCi n-[5,6-³H]hexanoic acid (American Radiolabeled Chemicals Inc., Cat# ART 1754-250) and 1 mM carnitine for 2 hours, maintaining serum-containing or serum-free conditions. The medium was collected and eluted in columns packed with DOWEX 1X2-400 ion exchange resin (Sigma-Aldrich, Cat# 217395) to analyze the released ³H₂O. Counts per min (CPM) were normalized to protein content in cell lysates. For cells treated with S63845 (Active Biochem, Cat# A-6044), the compound was added to the cells with [9,10(n)-³H]palmitic acid for two hours at a final sub-cytotoxic concentration of 1 μM. For tamoxifen-induced deletion of Mcl-1, cells were treated with tamoxifen or vehicle for 48 hours and allowed to recover for an additional 48 hours prior to conducting the experiment.

For FAO assays, cells in 6 well-plates were treated with 100μM etomoxir as indicated. Pulsing was performed in serum-free medium containing 1mM carnitine with 0.75 μCi [9,10(n)-3H] palmitic acid (GE Healthcare) for 2 hours. The medium was collected and eluted in columns packed with DOWEX 1X2-400 ion exchange resin (Sigma) to analyze the released ³H2O. ³H2O was measured in counts per minute (CPM) and normalized to total cellular protein using BCA Protein Assay Kit (ThermoFisher Scientific).