Cholesterol depletion and enrichment

Cholesterol was depleted using Methyl-β-cyclodextrin (MβCD; Sigma-Aldrich, St. Louis, MO). MβCD solution was prepared in PBS, and varying concentrations of MβCD were used. The desired concentration of MβCD solution was mixed with the cell suspension, and incubated for 30 min at 37°C and 5% CO₂. For cholesterol enrichment, MβCD-cholesterol complex (Sigma-Aldrich) was used with a cholesterol concentration of 40 mg/gm. The desired concentration of MβCD-cholesterol solution was mixed with the cell suspension, and incubated for 30 min at 37°C and 5% CO₂.

Cholesterol content in the cells was measured using an Amplex Red Cholesterol Assay Kit (Life Technologies), as per the manufacturer’s protocol. Approximately 0.75 × 10⁵ cells were used per well of a 96-well plate (Corning, Corning, NY). The fluorescence intensity was measured at 540 nm (excitation)/600 nm (emission) (Synergy2; Biotek, Winooski, VT). For assessment of the total cellular content, the cells were lysed by sonication. The cells were centrifuged at 300 × g for 10 min at room temperature; the pellets were resuspended in PBS, and the solution was vortexed for 60 s. The centrifugation and vortex steps were performed three times The pellet was then resuspended in PBS, sonicated for 90 s, centrifuged at 300 × g for 10 min at 4°C, resuspended in PBS, and vortexed for 45 s. The sonication with the accompanying steps was performed three times. The cell lysate was then tested for cholesterol content. For the plasma membrane cholesterol estimation, the cells were left intact. The cholesterol content was determined from a standard curve using 1–20 μg/mL cholesterol standards (the cholesterol standard provided in the kit was used as reference to obtain the standard curves; individual standard curves were obtained for each experiment). The fluorescence measurements were obtained using the automatic-gain feature, allowing the instrument software to select the best gain for a given run.