443 - The Measurement of Cellular Bioenergetics at Defined Steady-State Oxygen Concentrations: A Novel Microplate Based Method

Abstract

Although often overlooked, most cell-based in vitro assays are conducted under hyperoxic conditions, while the impact of oxygen concentration ([O2]) on experimental outcome is typically ignored. It is known however that for cells cultured at lower than ambient [O2], there exists a potential for higher [O2] to significantly impact cellular bioenergetics and, by extension, data output. While there is a growing appreciation of this deficiency, technical limitations have prevented broad uptake of [O2]-informed in vitro assay design. These limitations have been ameliorated by recent the advances in both instrumentation and assay technologies: integrated atmospheric control units (ACUs) now facilitate measurement at defined [O2] while advanced phosphorescent intracellular O2 and fluorescent extracellular pH probes, allowing simultaneous multiparametric measurements of cellular oxygenation, electron transport chain activity and glycolytic flux. Described herein is a high-throughput method for measuring all three key metabolic parameters under defined [O2] thereby further addressing such technical limitations. The method utilizes open-flow respirometry, a technique that allows for the measurement of cellular respiration at steady state (e.g. O2 supply = O2 demand). Preliminary data on respiration rate and media acidification at predefined [O2] is presented, facilitating a more detailed interrogatioen for the effect of [O2] on the balance between oxidative phosphorylation and glycolytic activity. This is further delineated using known metabolic effectors including the mitochondrial modulators, oligomycin, antimycin A and FCCP and the CPT-1 inhibitor Etomoxir. The method therefore complements existing technologies, and as a cost-effective, efficient means to measure cellular bioenergetics at an [O2] most relevant to a given cell type or experimental model.