Fig. 2.

Dependence on plasma membrane cholesterol concentration of cholesterol susceptibility to cholesterol oxidase. Fibroblasts were grown overnight in 96-well plates, and plasma membrane cholesterol was then adjusted as described for Fig. 1. The cells were washed and treated with 0.9 units of cholesterol oxidase in PBS for 10 min at 37°C. The H₂O₂ reaction product in aliquots of the overlying medium was determined with Amplex Red. Cholesterol remaining in the cells was determined by adding 0.2% Triton X-100 to the wells and allowing the oxidation reaction to run to completion before assay with Amplex Red. Values for the initial rate of cell cholesterol oxidation are averages of six replicate wells in each of eight experiments; they were plotted against relative cell cholesterol, and the curve was fit by eye to two straight lines.