Intracellular cholesterol quantitation kit

The intracellular total and free cholesterol were measured by an
Intracellular Cholesterol Quantitation Kit (Sigma-aldrich) according to the
manufacturer’s instructions. Briefly, after experimental treatment for
24 h, 2x10⁶ cells were trypsinized with Trypsin-EDTA followed by
centrifugation. Then the cell pellets were resuspended in a mix of chloroform:
isoporpanol: IGEPAL CA-630 (7:11:0.1). The samples were centrifuged at 13,000 g
for 10 min to remove insoluble material. Then the organic phase was transferred
to new tubes and air dried at 50°C for 30 min to remove chloroform,
followed by blowing the samples with nitrogen to remove any residual organic
solvent. Then the lipid was dissolved with 200 μl Cholesterol Assay
Buffer and 50 ul of the Reaction Mix was added to each standard and sample well
in a 96-well plate, and incubated for 60 min at 37°C in dark. The
absorbance was measured with a Spectra Max M5 Microplate Reader (Molecular
Devices) at OD 570 nm. Concentrations of cholesterol were calculated by dividing
the amount of cholesterol by the sample volume.

The liver cholesterol content was detected by an Intracellular Cholesterol Quantitation Kit (Sigma-Aldrich, St-Louis, MO) according to the manufacturer’s instruction. Briefly, liver tissue was weighed and then homogenized followed by being pretreated in the mix of chloroform:isoporpanol:IGEPAL CA-630 (7:11:0.1) at room temperature for 10 min. The samples were centrifuged at 13,000 g for 10 min to remove insoluble material. Then the organic phase was transferred to new tubes and air dried at 50°C for 30 min to remove chloroform, followed by blowing the samples with nitrogen to remove any residue organic solvent. After dried, the lipid was dissolved with 200 µL of the Cholesterol Assay Buffer and then diluted into 30× by this buffer for each sample. Then, 50 µL of the reaction mix was added to each 50 µL standard and sample well in a 96-well plate, and incubated for 60 min at 37°C in dark. The absorbance was detected with a Spectra Max M5 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) at OD 570 nm. Concentration of cholesterol in sample was calculated by dividing the amount of cholesterol by the sample weight.