How Supraphysiological Oxygen Levels in Standard Cell Culture Affect Oxygen-Consuming Reactions

Figure 2

Cells produce more H₂O₂ at 18% versus 5% O₂, but this is prevented or ameliorated by selective NADPH oxidase 1/4 inhibitor GKT137831. (a) C2C12 mouse myoblasts, (b) PC-3 human prostate cancer cells, and (c) wild-type and NOX1/2/4 triple knockout mouse dermal fibroblast cell lines were grown at 18% O₂ in a humidified 37°C CO₂ incubator, at 5%, and assayed at either 5% or 18% O₂ as in Maddalena et al. [3]. H₂O₂ efflux from cells was measured using Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine). Krebs-Ringer buffer (KRB) was used for the assays which consisted of 135 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 0.4 mM K₂HPO₄, 20 mM HEPES, 5.5 mM glucose, and 10% fetal bovine serum. During the experiment, cells were incubated in KRB-contained 50 mM Amplex Red reagent and 0.1 units/mL horseradish peroxidase enzyme in the presence of 5 μM GKT 137831 or vehicle control. Data were analysed using two-tailed t-tests. Bars represent the mean ± SEM from at least five independent experiments. .

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