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Optimization of a Multi-Mode Detection Model for Measuring Real-time Cellular Respiration and Mitochondrial Function using Fluorophoric Biosensors

Cellular metabolism characterization 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 timeresolved fluorescent mode is highlighted for generating drug compound dose response against OCR (μs/hr), presenting accurate comparisons of acidification rates converted to hydrogen ion scale (ECA[H+]/t), and converting lifetime detection to intracellular oxygen (% O2) in parallel with fluorescent imaging in live cell 2D monolayers.

Assay Overview and Detection Principle

The MitoXpress® Xtra – Extracellular Oxygen Consumption Assay [HS Method], pH-Xtra™ Glycolysis Assay, and MitoXpress® Intra – Intracellular O2 Assay are a family of fluorescent probes designed by Luxcel Biosciences to aide in the study of real-time analysis of mitochondrial function, metabolism and toxicity in a variety of biological matrices. The probes are chemically stable and inert, water-soluble, and can be multiplexed. The amount of fluorescent signal is an inverse relationship to intra- or extracellular O2 or proportional to extracellular H+ in the sample. Intracellular O2 levels (% O2), extracellular Oxygen Consumption Rate (OCR), and quantification of H+ levels are calculated from the changes in fluorescence signal over time. Probes can be optimized using a validation protocol in the kit insert for wet testing. Optimal detection parameters can then be applied to verification of cell lines and cell seeding densities using compound controls. Independent testing should be conducted to optimize each cell line individually.