Analysis of Intracellular Oxygen and Metabolic Responses of Mammalian Cells by Time-Resolved Fluorometry
- 15 November 2007
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 79 (24), 9414-9419
- https://doi.org/10.1021/ac701770b
Abstract
A simple, minimally invasive methodology for the analysis of intracellular oxygen in populations of live mammalian cells is described. Loading of the cells with the phosphorescent O(2)-sensing probe, MitoXpress, is achieved by passive liposomal transfer or facilitated endocytosis, followed by monitoring in standard microwell plates on a time-resolved fluorescent reader. Phosphorescence lifetime measurements provide accurate, real-time, quantitative assessment of average oxygen levels in resting cells and their alterations in response to stimulation. Analytical performance of the method is examined, optimized, and then demonstrated with different suspension and adherent cell lines including Jurkat, PC12, A549, HeLa, SH-SY5Y, and C2C12, by monitoring responses to mitochondrial uncouplers, inhibitors, plasma membrane depolarization, and Ca(2+) effectors. The assay provides relevant, information-rich data on cellular function and metabolism. It allows monitoring of small, rapid, and transient changes in cell respiration and screening of new chemical entities.This publication has 15 references indexed in Scilit:
- Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imagingAmerican Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 2007
- Three Distinct Mechanisms Generate Oxygen Free Radicals in Neurons and Contribute to Cell Death during Anoxia and ReoxygenationJournal of Neuroscience, 2007
- Circumventing the Crabtree Effect: Replacing Media Glucose with Galactose Increases Susceptibility of HepG2 Cells to Mitochondrial ToxicantsToxicological Sciences, 2007
- Analysis of mitochondrial function using phosphorescent oxygen-sensitive probesNature Protocols, 2006
- Oxygen Distribution and Consumption in the Developing Rat RetinaInvestigative Ophthalmology & Visual Science, 2006
- Mitochondria and calcium signalingCell Calcium, 2005
- Mitochondria in health and disease: perspectives on a new mitochondrial biologyMolecular Aspects of Medicine, 2004
- The mitochondrial calcium uniporter is a highly selective ion channelNature, 2004
- Redistribution of Intracellular Oxygen in Hypoxia by Nitric Oxide: Effect on HIF1αScience, 2003
- Does nitric oxide modulate mitochondrial energy generation and apoptosis?Nature Reviews Molecular Cell Biology, 2002