Cholesterol quantification via Amplex® Red assay

Membrane cholesterol levels were determined in cultured HUVEC after treatment with different CHIM-L concentrations (ranging from 0.1 to 10 µM). Quantification employed the commercial Amplex Red Cholesterol Kit (Invitrogen) according to the manufacturer’s instructions. Briefly, samples were diluted in reaction buffer and mixed with an equivalent volume of the Amplex Red working solution (300 µM Amplex Red, 2 U/ml horseradish peroxidase, 2 U/ml cholesterol oxidase and 0.2 U/ml cholesterol esterase). This resulted in a final concentration of 150 µM Amplex Red reagent in the reaction mix and a final volume of 100 µl per well. Samples were incubated for 90 min at 37 °C, subsequently washed three times, and fluorescence was determined by using a fluorescent microplate reader. All plates were protected from light and the data (n = 2) were plotted with Graphpad Prism 8.