Document Detail


Cholesterol interferes with the MTT assay in human epithelial-like (A549) and endothelial (HLMVE and HCAE) cells.
MedLine Citation:
PMID:  16510353     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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 A-1 decreased cholesterol-mediated inhibition of MTT assay. These studies indicate decreased MTT reduction as a result of enhanced exocytosis of formazan due to cholesterol. A careful validation of viability assay procedures is therefore suggested in experiments where cholesterol is a constituent, to avoid a potential bias in concluding results of cytotoxicity studies.
Authors:
Shama Ahmad; Aftab Ahmad; Kelly B Schneider; Carl W White
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  International journal of toxicology     Volume:  25     ISSN:  1091-5818     ISO Abbreviation:  Int. J. Toxicol.     Publication Date:    2006 Jan-Feb
Date Detail:
Created Date:  2006-03-02     Completed Date:  2006-04-25     Revised Date:  2006-10-30    
Medline Journal Info:
Nlm Unique ID:  9708436     Medline TA:  Int J Toxicol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  17-23     Citation Subset:  IM    
Affiliation:
Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, USA. ahmads@njc.org
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MeSH Terms
Descriptor/Qualifier:
Apolipoprotein A-I / pharmacology
Cell Line, Tumor
Cell Survival
Cholesterol / metabolism,  pharmacology*
Coloring Agents / analysis,  metabolism*
Dose-Response Relationship, Drug
Drug Interactions
Endothelium, Vascular / drug effects*,  metabolism
Epithelial Cells / drug effects*,  metabolism
Exocytosis / drug effects*
Humans
Oxidation-Reduction
Tetrazolium Salts / analysis,  metabolism*
Thiazoles / analysis,  metabolism*
Chemical
Reg. No./Substance:
0/Apolipoprotein A-I; 0/Coloring Agents; 0/Tetrazolium Salts; 0/Thiazoles; 298-93-1/thiazolyl blue; 57-88-5/Cholesterol

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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