Cholesterol cholesterol ester glotm assay

To determine intracellular cholesterol levels, a Cholesterol/Cholesterol Ester-Glo^TM Assay (Promega) was performed according to the manufacturer’s instructions. In brief, 20,000 cells were seeded per well in a 96-well plate in RPMI + 10% CS FCS. After 72 h of treatment, medium was removed and the cells were lysed with 50 µL Cholesterol Lysis Solution per well for 30 min at 37 °C. The cell lysis solution was then transferred into another 96-well plate (Corning 3610, white, clear bottom) and mixed with Cholesterol Detection Reagent in the presence of esterase. After incubation for 1 h at room temperature, chemoluminescence was measured on a Cytation™ 5 Cell Imaging Multi-Mode Reader (BioTek Instruments). Total cholesterol levels were normalized to protein content that was determined by Pierce^TM BCA protein assay (Thermo Fisher Scientific) and expressed as µM cholesterol.

According to the manufacturer’s protocol (Promega, Cholesterol/Cholesterol Ester-Glo^TM Assay), cells were seeded in a 96-well plate. The next day, the cells were counted and incubated with 50 μL of Cholesterol Lysis Solution for 30 min at 37 °C. Subsequently, 50 μL of Cholesterol Detection Reagent was added. We calculated the amount of free cholesterol by comparison of the luminescence of samples and standards.

Adrenal glands were dissected, clear of the surrounding fat pad, and homogenized in radioimmunoprecipitation assay buffer (Pierce, cat. no. 89900) supplemented with a protease and phosphatase inhibitor by Thermo Scientific (cat. no. A32961) at 4 °C, using lysing matrix tubes (MP Biomedicals, cat. no. 6913100) and a Bead Mill Homogenizer by Omni International. Tissue lysates were incubated for 1 hour in ice and spun down on a bench centrifuge for 20 minutes at 4 °C to get rid of unprocessed debris. Quantification of cholesterol was carried out using a Cholesterol/Cholesterol Ester-Glo^TM Assay by Promega (cat. no. J3190) following the manufacturer's instructions, with the exception that adrenal lysates were diluted from 1:10 to 1:40 in the lysis buffer provided by the kit to fit the calibration curve. The assay was performed either with or without cholesterol esterase, to allow for quantification of both total and free cholesterol, respectively. Values for esterified cholesterol were obtained by subtraction of free from total cholesterol. All cholesterol values were normalized by protein concentration assayed using a DC protein assay (Bio-Rad, cat. no. 5000112).