title: High cell density attenuates reactive oxygen species: implications for in vitro assays.
abstract: In vitro cell-based assays are an essential and universally used step in elucidation of biological processes as well as in drug development. However, results obtained depend on the validity of protocols used. This statement certainly pertains to in vitro assays of oxidative stress. The holy grail of in vitro models is reliability and predictability of outcomes that relate to a single variable like addition of hydrogen peroxide or xanthine oxidase. Without such validated outcomes, comparison of results among different laboratories is not possible. Achieving this goal requires a thorough understanding of the complex interplay between the cells, their environment, and the experimental assays. Furthermore, as this knowledge is attained, it must be disseminated and used to update and standardize existing protocols. Here, we confirm and extend the effect of pyruvate and cell density on in vitro oxidative stress assays. Cell viability was assessed using a colorimetric assay measuring the reduction of a tetrazolium salt (XTT) into a colored formazan dye. Extracellular hydrogen peroxide concentrations were measured using the foxp3 assay. We confirmed a previously reported finding that pyruvate, a common ingredient in cell culture media, acts as an extracellular scavenger of reactive oxygen species. We also demonstrated that cell density directly correlates with resistance to oxidative stress in tissue culture. It is theorized that the protective effect due to cell density predominantly relates to intracellular factors such as reduced glutathione and extracellular factors such as catalase.
authors: School of Medicine, Stony Brook University, Stony Brook, New York, USA
authors: Dennis P
authors: DP
authors: Kim
authors: Jonathan
authors: J
authors: Yahav
authors: Michael
authors: M
authors: Sperandeo
authors: Lauren
authors: L
authors: Maloney
authors: Monica
authors: M
authors: McTigue
authors: Fubao
authors: F
authors: Lin
authors: Richard A F
authors: RA
authors: Clark
mesh_terms: D002452:Cell Count; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003124:Colorimetry; D005110:Extracellular Space / Q000737:chemistry; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D051858:Forkhead Transcription Factors / Q000032:analysis; D005562:Formazans / Q000032:analysis; D016166:Free Radical Scavengers / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis / Q000378:metabolism*; D007202:Indicators and Reagents / Q000032:analysis; D018384:Oxidative Stress / Q000187:drug effects*; D019289:Pyruvic Acid / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015203:Reproducibility of Results; D013778:Tetrazolium Salts / Q000032:analysis* / Q000378:metabolism
chemical_list: C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D005562:Formazans; D016166:Free Radical Scavengers; D007202:Indicators and Reagents; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; D019289:Pyruvic Acid; D006861:Hydrogen Peroxide