Wendy Goodrich, BioTek Instruments, Inc. Winooski, VT USA

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Introduction

Characterization of cellular metabolism is being aided by the development of new tools designed to provide ease-of-use, higher throughput, and multiplexed data markers for analysis. One of these tools is a simple mix and measure assay compatible with a variety of cellular matrices that utilizes fluorophoric probes to measure oxygen consumption rates (OCR), extracellular acidification (ECA), and intracellular oxygen levels useful to inform on the activity of the electron transport chain (ETC) and glycolytic flux. These probes can be detected using standard fluorescence, time-resolved fluorescence, or lifetime fluorescence with reduced background and increased signal dynamic range dependent on the detection mode. Optimization of biosensor recognition in all three fluorescent modes was done in microplate format using multiple cell lines and drug compound treatments. In particular, the lifetime time-resolved fluorescent mode is highlighted for generating drug compound dose response against OCR (&mirco;s/hr), presenting accurate comparisons of acidification rates converted to hydrogen ion scale (ECA[H+]/t), and detecting intracellular oxygen levels in parallel with fluorescent imaging in live cell 2D monolayers.