Quantifying Mitochondrial Phospholipids

Levels of phospholipids in mitochondria were measured with a phospholipid assay kit (MilliporeSigma; catalog number MAK122) as described previously (Sun et al., 2021 (link)). Briefly, fractions of mitochondria were diluted to 1–2.5 μg protein per assay using the assay buffer provided. Each reaction mix was set up by adding a prepared sample or standard to phospholipid D that degrades phospholipids to release choline. The amount of choline was determined with choline oxidase and an H₂O₂ specific dye. A colorimetric reading at wavelength 570 nm was proportional to the phospholipid concentration in the sample. Results were calculated according to the standard curve and normalized by protein amount per sample, and measurements were performed in triplicates.