Fluoresterol

Guide (The following is our recommended solution. This solution is merely a guideline and should be modified according to your specific needs.)
By detecting the fluorescence signal of 22-NBD Cholesterol (Fluoresterol) to reflect the cholesterol efflux efficiency, in vitro fluorescence labeling and cholesterol efflux experiments^[1]: 1. Cell preparation: Mouse macrophage RAW264.7 cells were seeded at a density of 3×10⁴ cells per well in a 96-well plate. 1% penicillin-streptomycin and 10% bovine serum-containing DMEM medium was added, and the cells were incubated in a 37°C, 5% CO₂ incubator for 16 hours. Mouse peritoneal macrophages were induced with 4% thioacetamide solution and then cultured on a 100% DMEM medium for 2 hours before being transferred to a new culture medium for another 48 hours for later use.
2. Fluorescence labeling: 0.2 mL of the labeled medium containing methyl-β-cyclodextrin, Topflour cholesterol (a fluorescent cholesterol analog) and unlabeled cholesterol was added to the RAW264.7 cells. After incubation for 6 hours, the cells were washed with DMEM medium to remove the unbound fluorescent cholesterol; the mouse peritoneal macrophages were treated using the same labeling method.
3. Drug treatment and incubation: After equilibrating the cells with DMEM medium containing 0.2% BSA for 1 hour, add DMEM medium containing apolipoprotein A1 (apoA1, 10 μg/mL), along with different concentration gradients of ethanol extract of Dianliangpi (EEDL: 100 μg/mL, 200 μg/mL, 400 μg/mL) or LXR agonist T0901317 (0.2 μM, 0.5 μM), and incubate at 37°C and 5% CO₂ for 18 hours.
4. Fluorescence Detection: Collect the cell culture supernatant, add lysis buffer to the cell layer, and incubate at room temperature for 30 minutes to prepare the cell lysate. Use an enzyme reader to detect the fluorescence intensity of the supernatant and the lysate. The excitation wavelength is 482 nm and the emission wavelength is 516 nm. Calculate the cholesterol efflux efficiency.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.