Mitophagy Imaging in MEFs

MEFs were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin at 37°C and 5% CO2. Glucose and acetoacetate containing media were made as previously described (Mishra et al., 2014 (link)). For mitophagy experiments, cells were plated on Nunc Lab-Tek II Chambered Coverglass slides (155409, Thermo) in DMEM-based media. After cells had adhered, they were washed with PBS and glucose- or acetoacetate-containing medium was applied, after which cells were allowed to grow for 4 days and then imaged. Because cells grow more slowly in acetoacetate medium, a four-fold excess of cells was plated relative to glucose medium so that both samples were at the same density on the day of imaging.