The picomole determination of free and total cholesterol in cells in culture.
An enzymatic, fluorometric method for the determination of free and total cholesterol in cells in culture is presented. The method is simple, requiring one reagent that includes all of the enzymes and a second reagent that increases the pH, which enhances the fluorescence of the product. The method is based on the enzymatic hydrolysis of cholesteryl esters to free cholesterol, the oxidation of cholesterol with the liberation of hydrogen peroxide, and the reaction of this peroxide with a fluorogen to form a fluoresencet product in the presence of a peroxidase. It is rapid, in that free cholesterol can be read in 5 minutes and total cholesterol after 20 minutes. The precision of the method is greater than that obtained from gas-liquid chromatography.
D000818:Animals; D002478:Cells, Cultured; D002784:Cholesterol / Q000032:analysis*; D002788:Cholesterol Esters / Q000032:analysis*; D000882:Haplorhini; D008832:Microchemistry; D013050:Spectrometry, Fluorescence / Q000379:methods
D002788:Cholesterol Esters; D002784:Cholesterol