There is a need for accurate, high-throughput measures to gauge the efficacy of potential drugs in living cells. Metabolism is an early marker of drug response in cells, and NADH and FAD are autofluorescent cellular metabolic coenzymes that can be non-invasively monitored using optical techniques. Relative rates of glycolysis and oxidative phosphorylation in a cell are quantified by the redox ratio, defined as the autofluorescence intensity of NADH divided by that of FAD. Microplate readers are high-throughput instruments that can rapidly measure NADH and FAD autofluorescence intensities for hundreds of wells, and are capable of identifying receptor status and resolving drug response in breast cancer cell lines. This study tests the accuracy and repeatability of plate reader experiments measuring the redox ratio in breast cancer cell lines. NADH and FAD fluorescence levels remained constant over the course of multiple measurements (p<0.1), ruling out the incidence of photobleaching. The contribution of media to background fluorescence signal was also investigated. Media fluorescence levels for both coenzymes were significantly lower (p<0.0001) than those from wells containing cells, and replacing the media with saline resulted in the same redox ratio trends among cell lines as initial measurements with media. Following treatment with carbonyl cyanide p-fluorodeoxyphenylhydrazone (FCCP), an oxidative phosphorylation inhibitor, the redox ratio decreased (p<0.05), validating NADH and FAD as the primary fluorescence sources. These findings verify that autofluorescence measurements taken by microplate readers accurately and reliably characterize NADH and FAD fluorescence, validating their promise in the areas of metabolic monitoring and drug development.

Date Published: 26 February 2015
PDF: 6 pages
Proc. SPIE 9303, Photonic Therapeutics and Diagnostics XI, 93032S (26 February 2015); doi: 10.1117/12.2077941

Published in SPIE Proceedings Vol. 9303:
Photonic Therapeutics and Diagnostics XI
Hyun Wook Kang; Brian J. F. Wong M.D.; Melissa C. Skala; Bernard Choi; Guillermo J. Tearney M.D.; Andreas Mandelis; Nikiforos Kollias; Kenton W. Gregory M.D.; Mark W. Dewhirst D.V.M.; Justus F. Ilgner M.D.; Alfred Nuttal; Haishan Zeng; Laura Marcu; Claus-Peter Richter, Editor(s)