Abstract 4844: Development of an oxygen consumption rate assay for a standard plate reader

Abstract

Abstract Cells generate ATP by two metabolic pathways, oxidative phosphorylation (OXPHOS) and glycolysis. Together, the amount of ATP generated by both OXPHOS and glycolysis define a cell’s metabolic phenotype and fitness. Researchers are interested in methods to characterize cellular metabolic fitness, which can be a powerful a selection criterion for cell-based therapies such as CAR-T cell therapy. Researchers are also interested in measuring the effects of drug treatment on metabolic fitness, to identify metabolic targets for cancer therapeutics and to determine mitochondrial toxicity of novel therapeutics. Here, we have developed a low-cost strategy for a standard plate reader to monitor the rate change of oxygen concentration, or the oxygen consumption rate (OCR), which is a measure of OXPHOS activity. In our assay, oxygen quenches a phosphorescent porphyrin dye in a concentration dependent manner, allowing one to calculate the oxygen concentration of the culture system with the Stern-Volmer relationship. The porphyrin dye is embedded within nanobeads that are suspended above the cell monolayer and the phosphorescence of the beads is measured from the bottom of the well with a standard plate reader. We found that the OCR increased with cell seeding density in three separate cell lines: HEP-G2, HCT-116, and K-562 suspension cells. We showed that inhibitors of the electron transport chain (ETC) decreased OCR, whereas an uncoupler of the electron transport chain, FCCP, increased OCR in a dose dependent manner. Lastly, mitochondrial fitness (e.g., spare respiratory capacity) was determined by sequentially dosing the mitochondria with poisons specific to the different ETC complexes. Citation Format: Ian Marozas, Zhiyang Zeng, Mike Valley, Jolanta Vidugiriene, James Cali, Wenhui Zhou. Development of an oxygen consumption rate assay for a standard plate reader. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4844.