Integration of high-content fluorescence imaging into the metabolic flux assay reveals insights into mitochondrial properties and functions
| Autor: | Vinee Purohit, Sofia D. Merajver, Hanna S. Hong, Joel A. Yates, Samuel A. Kerk, Andrew C. Little, Ilya Kovalenko, David B. Lombard, Costas A. Lyssiotis, Laura E. Goo |
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| Rok vydání: | 2019 |
| Předmět: |
chemistry.chemical_classification
Membrane potential 0303 health sciences Reactive oxygen species Complete data Fluorescence-lifetime imaging microscopy Standard technique Fluorescence 03 medical and health sciences 0302 clinical medicine chemistry 030220 oncology & carcinogenesis Respiration Biophysics Glycolysis 030304 developmental biology |
| DOI: | 10.1101/758110 |
| Popis: | SUMMARYMetabolic flux technology with the Seahorse bioanalyzer has emerged as a standard technique in cellular metabolism studies, allowing for simultaneous kinetic measurements of respiration and glycolysis. Methods to extend the utility and versatility of the metabolic flux assay would undoubtedly have immediate and wide-reaching impacts. Herein, we describe a platform that couples the metabolic flux assay with high-content fluorescence imaging to simultaneously enhance normalization of respiration data with cell number; analyze cell cycle progression; quantify mitochondrial content, fragmentation state, membrane potential, and mitochondrial reactive oxygen species. Integration of fluorescent dyes directly into the metabolic flux assay generates a more complete data set of mitochondrial features in a single assay. Moreover, application of this integrated strategy revealed insights into mitochondrial function following PGC1a and PRC1 inhibition in pancreatic cancer and demonstrated how the Rho-GTPases impact mitochondrial dynamics in breast cancer. |
| Databáze: | OpenAIRE |
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