The use of a fluorescence-based oxygen uptake assay in the analysis of cytotoxicity.
Cellular oxygen uptake is an informative parameter of cellular function but is not measured routinely in the analysis of cytotoxicity. Here we have evaluated the ability of a fluorescence-based oxygen uptake assay to assess the metabolic activity of common adherent cells including HepG2, LLC-PK1, Hek293T, C2C12, H-4-II-E, and primary rat hepatocytes. The assay employs water-soluble phosphorescent oxygen probes, analysed in standard 96-well plates on a conventional fluorescence plate reader. Using this respirometric method, cellular responses to known toxicants were examined and results compared to those obtained using established cell viability assays such as MTT, LDH and CyQuant. Respirometric analysis successfully detected these cytotoxic insults with responses being influenced by both mode of toxicity and the biochemical characteristics of the individual cell line. Results indicate that the oxygen uptake assay was more sensitive to the impairment of mitochondrial function than the other assays used. In conjunction with assays analysing other biomarkers of cytotoxicity, a more detailed picture of cell response to drug treatment can be obtained.
Hynes
James
J
Biochemistry Department, University College Cork, Lee Maltings, Cork, Ireland.
Hill
Roger
R
Papkovsky
Dmitri B
DB
D000818:Animals; D002448:Cell Adhesion; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D005453:Fluorescence; D006801:Humans; D007654:Ketoconazole / Q000633:toxicity; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010101:Oxygen Consumption / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018675:Toxicity Tests
D007654:Ketoconazole