TY - JOUR
T1 - High-content fluorescence imaging with the metabolic flux assay reveals insights into mitochondrial properties and functions
AU - Little, Andrew Charles
AU - Kovalenko, Ilya
AU - Goo, Laura Elaine
AU - Hong, Hanna Sungok
AU - Kerk, Samuel Andrew
AU - Yates, Joel Anthony
AU - Purohit, Vinee
AU - Lombard, David Benner
AU - Merajver, Sofia Diana
AU - Lyssiotis, Costas Andreas
N1 - Funding Information:
We would like to thank Dr. John Dishinger of BioTek for his assistance with designing imaging and image analysis protocols. We wish to thank Prof. Youle RJ for providing the YFP-Parkin plasmid and Dr. Stefan Prechtl and Dr. Sven Christian for initial proof-of-concept experiments. Fig. 1 and Supplementary Figure 1 were created using BioRender. com. C.A.L. was supported by a Pancreatic Cancer Action Network/AACR Pathway to Leadership award (13-70-25-LYSS); Junior Scholar Award from The V Foundation for Cancer Research (V2016-009); Kimmel Scholar Award from the Sidney Kimmel Foundation for Cancer Research (SKF-16-005); a 2017 AACR NextGen Grant for Transformative Cancer Research (17-20-01-LYSS); an ACS Research Scholar Grant (RSG-18-186-01); NIH-R37-CA237421 and NIH-R01-CA248160. D.L. was supported by R01GM101171. A.C.L. and S.D.M. were supported by the Breast Cancer Research Foundation. C.A.L. and S.D.M. were supported by the Rogel Cancer Center core grant NIH-P30-CA046592-29.
Publisher Copyright:
© 2020, The Author(s).
PY - 2020/12/1
Y1 - 2020/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s42003-020-0988-z
DO - 10.1038/s42003-020-0988-z
M3 - Article
C2 - 32472013
AN - SCOPUS:85085699075
SN - 2399-3642
VL - 3
JO - Communications Biology
JF - Communications Biology
IS - 1
M1 - 271
ER -