High-content fluorescence imaging with the metabolic flux assay reveals insights into mitochondrial properties and functions

Andrew Charles Little, Ilya Kovalenko, Laura Elaine Goo, Hanna Sungok Hong, Samuel Andrew Kerk, Joel Anthony Yates, Vinee Purohit, David Benner Lombard, Sofia Diana Merajver, Costas Andreas Lyssiotis

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

Metabolic 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 provide means for normalization of respiration data with cell number; analyze cell cycle distribution; and 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.

Original languageEnglish (US)
Article number271
JournalCommunications Biology
Volume3
Issue number1
DOIs
StatePublished - Dec 1 2020

All Science Journal Classification (ASJC) codes

  • Medicine (miscellaneous)
  • General Biochemistry, Genetics and Molecular Biology
  • General Agricultural and Biological Sciences

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