Department of Internal Medicine, Division of Hematology and Oncology, University of Michigan, Ann Arbor, MI, 48109, USA.
Rogel Cancer Center, University of Michigan, Ann Arbor, MI, 48109, USA.
Commun Biol. 2020 May 29;3(1):271. doi: 10.1038/s42003-020-0988-z.
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.
基于 Seahorse 生物分析仪的代谢通量技术已经成为细胞代谢研究中的标准技术，可实现呼吸和糖酵解的动力学同时测量。扩展代谢通量测定法的用途和多功能性的方法无疑会产生直接而广泛的影响。在此，我们描述了一个将代谢通量测定与高内涵荧光成像相结合的平台，该平台可同时提供呼吸数据与细胞数量归一化、分析细胞周期分布以及定量线粒体含量、片段状态、膜电位和线粒体活性氧的方法。荧光染料直接整合到代谢通量测定中，可在单次测定中生成更完整的线粒体特征数据集。此外，该集成策略的应用揭示了 PGC1a 和 PRC1 抑制对胰腺癌中线粒体功能的影响，并展示了 Rho-GTPases 如何影响乳腺癌中线粒体动力学。
