C7510

For all experiments, RBC membranes (pink ghosts) were prepared according to Hanahan et al^{10 (link)}; in brief, blood was centrifuged at 1000g for 30 minutes at 4°C, plasma and buffy coat were removed, and RBCs were suspended in 1 mL of 310 mOsm/L (0.172 mol/L) Tris-HCl buffer (pH 7.6). Samples were washed twice with 310 mOsm/L Tris-HCl buffer and resuspended to a final hematocrit of 50%. One mL of RBC suspension was pelleted; RBCs were resuspended in 1 mL of hypotonic (20 mOsm/L) Tris-HCl buffer (pH 7.6) and lysed on ice for 5 minutes. RBC membranes were centrifuged at 20 000g for 40 minutes at 4°C, washed 4 times, and resuspended in 20 mOsm/L Tris-HCl buffer to a final volume of 1 mL. Colorimetric assay was performed in 96-well plates; samples were prepared by combining 36 μL of RBC membrane suspensions with 324 μL of Infinity cholesterol reagent (Thermo Scientific catalog no. TR13521), and incubating the mixtures for 1 hour at 37°C. Plates were read at 500 nm; commercial cholesterol preparation (Pointe Scientific, Core Laboratory Supplies catalog no. C7510) was used to derive a standard curve.