Quantifying Cellular Cholesterol in Cell Lines

To quantify cellular cholesterol in HeLa and A549 cells, 1.5 × 10⁵ cells per well were seeded on 6-well culture plates in complete medium for 24 h. Cells were treated in serum-free medium for a further 24 h with vehicle, 10 µM dexamethasone, 10 µM hydrocortisone or 1 mM methyl-β-cyclodextrin, before the cells were washed twice with PBS, collected in 200 µl/ well cholesterol assay buffer (Thermo Fisher Scientific) and stored at −20°C. Cellular cholesterol was measured using the Amplex Red Cholesterol Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, the assay uses cholesterol esterase from Pseudomonas to hydrolyze cholesterol esters before cholesterol oxidase from Streptomyces oxidizes cellular cholesterol to yield hydrogen peroxide and cholestenone. The Amplex Red reagent reacts with hydrogen peroxide in the presence of horseradish peroxidase to produce fluorescent resorufin. Fluorescence was measured by a POLARstar Omega microplate reader using 530 nm excitation and 590 nm emission. Protein abundance was measured in samples using a DC protein assay (Bio-Rad, Hercules, CA, United States), and cholesterol concentrations were normalized to total cellular protein. The inter- and intra-assay coefficients of variation for the assay were < 6% and < 5% respectively.