Cholesterol interferes with the MTT assay in human epithelial-like (A549) and endothelial (HLMVE and HCAE) cells

International Journal of Toxicology
Shama AhmadCarl W White

Abstract

Metabolically active cells are able to convert the MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye to blue formazan. This is the basis of the MTT assay, which is among the most widely used screening methods to evaluate cell viability and proliferation. When testing the effects of cholesterol products on the viability of human pulmonary epithelial-like A549 cells using trypan blue staining (cell numbers) and the MTT assay, results were inconsistent. The MTT assay indicated greater than 50% loss of viability with exposure of cells to cholesterol, whereas there was no decrease in viability indicated by trypan blue exclusion and propidium iodide uptake. A similar decrease in MTT reduction was obtained upon cholesterol treatment in human lung microvascular endothelial cells (HLMVECs) and human coronary artery endothelial cells (HCAECs) without loss of viability. This suggested a direct interference of cholesterol with the assay. However, using a cell-free system, there was no decrease in the reduction of MTT by ascorbic acid during incubation with a similar concentration of cholesterol. Light microscopy revealed enhanced exocytosis of formazan granules in presence of cholesterol. Incubation with apolipoprotein ...Continue Reading

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Methods Mentioned

BETA
transfection
phase-contrast
flow cytometry
light microscopy

Software Mentioned

JMP

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