Cholesterol assay buffer

The bovine endometrial stromal cells and HeLa cells were grown at a density of 10⁵ cells/well in 12-well tissue culture plates for 24 h in complete media, and then cultured for 24 h in serum-free media with or without 2 mM glutamine, or 0.5 mM methyl-β-cyclodextrin, as described in Results. After the treatment period, cells were collected in 200 μl/well cholesterol assay buffer (Thermo Fisher Scientific) and stored in Eppendorf tubes at -20°C. When needed, samples were defrosted at room temperature and sonicated for 10 min in a sonicating water bath. Cellular cholesterol content was measured using the Amplex® Red Cholesterol Assay Kit (Thermo Fisher Scientific). Total cellular phospholipid was measured in the samples prepared for the cholesterol assay using a phospholipid assay kit (MAK122, Sigma). Cholesterol concentrations were then normalized to phospholipid concentrations.

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.

To measure cellular cholesterol, 1.5 × 10⁵ HeLa cells/well were cultured in complete medium for 24 h using 6-well culture plates (TPP), and then treated in serum-free medium with 10 ng/ml 27-hydroxycholesterol, 10 ng/ml 25-hydroxycholesterol, 1 mM methyl-β-cyclodextrin (Merck) or 10 µM atorvastatin for 24 h. The cells were then washed twice with PBS, collected in 200 µl/well cholesterol assay buffer (Thermo Fisher Scientific), and stored at -20⁰C. Cellular cholesterol was measured using the Amplex Red Cholesterol Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cholesterol concentrations were normalized to total protein concentrations, measured using a DC protein assay (Bio-Rad Laboratories, Hercules, CA, United States), as described previously (18 (link), 29 (link)). The inter- and intra-assay coefficients of variation for the cholesterol assay were < 5% and < 6% respectively.