Isolation and Metabolic Analysis of RPE Cells

RPE cells from VMD2-Cre;Vhl^fl/fl and control Vhl^fl/fl littermates were isolated as previously described (Krohne et al., 2012 (link)). Cells were maintained at 37° and 5% CO₂ in DMEM/F12 from Thermo Fisher Scientific (Waltham, MA) with 2% FBS from Jackson ImmunoResearch (West Grove, PA). 2 μg/μl doxycycline was added to all cultures daily for three days to induce Vhl ablation. Human RPE cells (Lonza) were maintained either on plastic surfaces or on transwell filters (Corning) depending on the application in RtEGM Retinal Pigment Epithelial Cell Growth Medium fortified with RtEGM BulletKit from Lonza (Basel, Switzerland). Glucose, lactate, and glutamine levels were measured from the media in apical and basal compartments using a 2900 Biochemistry Analyzer from YSI (Yellow Springs, OH). A concentrated stock of DMOG from Cayman Chemical (Ann Arbor, MI) was made with DMSO, and added directly to the media in different concentrations to induce pseudo-hypoxia. RPE cells were maintained in low oxygen and metabolic changes were analyzed using Seahorse Flux Analysis (see below) in a Coy Dual Hypoxia Chamber from Coy Lab Products (Grass Lake, MI) (Grassian et al., 2014 (link)).