Cholesterol cholesterol ester glo assay

Plasma and retinas were collected from RA, OIR and K604-treated OIR pups at P17. The levels of cholesterol esters were measured using luminescence-based Cholesterol/Cholesterol Ester Glo™ Assay (J3190, Promega, Madison, WI) as per manufacturers’ protocol. Briefly, retinas were homogenized, and blood plasma were diluted (1:10) in Cholesterol lysis solution and incubated at 37 °C for 30 min. Equal amounts of extracts were incubated in cholesterol detection reagent with and without cholesterol esterase enzyme in a 96 well white bottom plates at room temperature for 1 h. Luminescence was recorded using Polar Star Omega microplate reader (BMG Labtech Inc, NC). Total and free cholesterol concentrations were measured by comparing the luminescence of samples with and without cholesterol esterase, respectively. Cholesterol ester concentrations were calculated as the difference between total and free cholesterol concentrations.

BNI274 was seeded in 96‐well plates in triplicate with 3000 cells per well. Vitamin D3 with the concentration of 5 μm or dimethylsulfoxide was added into the cell culture medium for 24‐h treatment. Then total cellular cholesterol level was measured using Cholesterol/Cholesterol Ester‐Glo Assay (Promega) in accordance with the manufacturer's instructions. The detection result was normalized to cell viability.

TG and TC content in steatotic HepG2 cells was determined with the Triglyceride-Glo Assay (#J3160, Promega) and Cholesterol/Cholesterol Ester-Glo Assay, (#J3190, Promega), respectively. Briefly, HepG2 cells that had been subjected to siRNA-mediated mRNA knockdown were seeded in a 96-well plate (3000 cells/well) and treated with 0.5 mM OA for 24 h. The medium was removed, and cells were washed twice by Hank’s balanced salt solution and lysed with 30 μl buffer included in the kits. The concentration of each of TG and TC was calculated based on standard curves. Mean and 95% CI values for each experimental group were calculated from five independent wells, and three independent experiments were performed. The statistical significance was determined using the Student’s t test.

Huh7 cells were plated at a density of 5000 cells/well in a 96 well plate in DMEM with 2% FBS. Cells were treated with DFMO for 96h or after 72h, treated with DEF or GC7 for 24h. The following day, the media was removed from cells followed by a PBS wash. To measure total intracellular cholesterol abundance, we used the Cholesterol/Cholesterol Ester-Glo Assay (Promega) in accordance to manufacturer’s protocol.

About 2 × 10⁵ cells were seeded in a 60 mm dish, and APC or other drugs were supplemented into the medium 36 h later. After about 12–24 h of treatment, the cells were harvested for enzyme-linked immunosorbent assay (ELISA) detection according to protocols supplied by the cAMP ELISA kit (L00460, GenScript, Piscataway, NJ, USA) and a PGE2 Express ELISA Kit (500141, Cayman, Michigan, USA), or for triglyceride and cholesterol detection according to protocols supplied by the Triglyceride-Glo™ Assay (J3160, Promega, Madison, WI, USA) and the Cholesterol/Cholesterol Ester-Glo™ Assay (J3190, Promega).