Cholesterol quantification assay kit

HeLa cells were plated at 80–90% confluency in 150 mm dishes and transfected with 5 µg of plasmid DNA encoding VPA0226-WT sf-GFPN1 or VPA0226-H/A sf-GFPN1 using Polyjet transfection reagent as described above. 14 hr post transfection, plasma membranes were isolated from the transfected cells using subcellular fractionation and differential centrifugation following previously published protocols (Tannu and Hemby, 2006 (link); Casey et al., 2017 (link)). Briefly, cells harvested by scraping into PBS were washed and re-suspended in HNMEK lysis buffer (20 mM HEPES pH 7.4, 50 mM NaCl, 2 mM MgCl2, 2 mM EDTA, 10 mM KCl, 50 nM EGTA, protease inhibitors) for 30 min on ice. The cell suspensions were lysed by dounce homogenizer and the homogenate was centrifuged at 800 g and 4°C for 10 min to obtain the nuclei pellet (P1). The supernatant was centrifuged at 10,000 g, 4°C for 15 min to pellet the organelle fraction (P2). The supernatant was further centrifuged at 100,000 g, 4°C for 1 hr to obtain the plasma membrane (P3) and cytosol (S3) fractions. All the pellets were washed once in HNMEK buffer and samples were collected for protein quantification and western blot analysis. Free cholesterol quantification in the plasma membrane fractions was carried out using a cholesterol quantification assay kit (Sigma, CS0005) following manufacturer’s instructions.

Cholesterol Quantification Assay Kit (Sigma-Aldrich, CS0005) was used to measure the cholesterol concentration of samples. Briefly, 44 μL Assay Buffer, 2 μL Probe, 2 μL Enzyme Mix, 2 μL Cholesterol Esterase, and 50 μL sample were mixed and incubated at 37 °C for 30 min in each well. A calibration curve was established for every measurement with standard samples with 0–5 μg cholesterol. All samples were diluted to the range of the calibration curve with the Assay Buffer. Absorbance at 570 nm was measured and compared to the standards on the same plate to determine total cholesterol.