HAP1 Cell Growth and Lipid Extraction

WT, COQ8A^KO, COQ8B^KO, and COQ8A/B^DKO HAP1 cells were obtained from Horizon Discovery and previously validated²⁶. Three 10 cm plates were seeded with 1.4×10⁶ cells for each cell line in IMDM (Thermo 12440053) supplemented with 10% heat inactivated FBS (HI FBS)(Atlanta Biologicals) and 100 U/mL Penicillin-Streptomycin (Pen/Strep)(Thermo 15140122). Cells were grown at 37 °C, 5% CO₂ for three days to approximately 90% confluency. Each plate of cells was washed with 5 mL dPBS then isolated by trypsinization. Cells were pelleted (173 g, 5 min, RT) and media was removed. Cells were then resuspended in 5 mL media and counted using the Muse Count and Viability Assay (Luminex). 1.2×10⁷ viable cells were collected per sample and pelleted by centrifugation (173 g, 5 min, RT). Media was removed and cells were resuspended in 5 mL dPBS. Cells were pelleted again by centrifugation and dPBS was removed. Cell pellets were resuspended in 600 μL cold methanol with 0.1 μM CoQ₈ (Avanti Lipids 900151p-1mg) as the internal standard. Suspended cells were transferred to screw cap tubes and vortexed for 10 min at 4 °C to lyse. 400 μL cold petroleum ether was added to each tube. Samples were vortexed for 3 min at 4 °C then centrifuged at 800 g, 3 min, RT to separate the layers. The top petroleum ether layer was collected in a new tube and the extraction was repeated with an additional 400 μL petroleum ether. The two extractions were combined, and the ether was removed under argon gas. Once dried, the lipids were resuspended in 100 μL methanol, transferred to glass autosampler vials, and submitted for LC-MS lipid measurements.