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METHODS article
Front. Cell Dev. Biol.
Sec. Signaling
Volume 12 - 2024 |
doi: 10.3389/fcell.2024.1460061
This article is part of the Research Topic Survival Strategies: Cellular Responses to Stress and Damage View all articles
Fast and quantitative mitophagy assessment by flow cytometry using the mito-QC reporter
Provisionally accepted- 1 Margarita Salas Center for Biological Research, Spanish National Research Council (CSIC), Madrid, Spain
- 2 Department of Neurosciences and Movement Science, Faculty of Science and Medicine, University of Fribourg, Fribourg, Fribourg, Switzerland
Mitochondrial quality control is finely tuned by mitophagy, the selective degradation of mitochondria through autophagy, and mitochondrial biogenesis. Removal of damaged mitochondria is essential to preserve cellular bioenergetics and prevent detrimental events such as sustained mitoROS production, pro-apoptotic cytochrome c release or mtDNA leakage. The array of tools available to study mitophagy is very limited but in constant development. Almost a decade ago, we developed a method to assess mitophagy flux using MitoTracker Deep Red in combination with lysosomal inhibitors. Now, using the novel tandem-fluorescence reporter mito-QC (mCherry-GFP-FIS1101-152) that allows to differentiate between healthy mitochondria (mCherry+GFP+) and mitolysosomes (mCherry+GFP-), we have developed a robust and quantitative method to assess mitophagy by flow cytometry. This approach has been validated in ARPE-19 cells using PINK1/Parkin-dependent (CCCP) and PINK1/Parkin-independent (DFP) positive controls and complementary techniques. Furthermore, we show that the mito-QC reporter can be multiplexed, especially if using spectral flow cytometry, to simultaneously study other cellular parameters such as viability or ROS production. Using this technique, we evaluated and characterized two prospective mitophagy inducers and further dissected their mechanism of action. Finally, using mito-QC reporter mice, we developed a protocol to measure mitophagy levels in the retina ex vivo. This novel methodology will propel mitophagy research forward and accelerate the discovery of novel mitophagy modulators.
Keywords: FACS, Mitochondria, Autophagy, Retina, SI, Fisetin, Phenanthroline
Received: 05 Jul 2024; Accepted: 02 Sep 2024.
Copyright: © 2024 Jiménez-Loygorri, Jiménez-García, Viedma-Poyatos and Boya. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Carlos Jiménez-García, Department of Neurosciences and Movement Science, Faculty of Science and Medicine, University of Fribourg, Fribourg, CH-1700, Fribourg, Switzerland
Álvaro Viedma-Poyatos, Margarita Salas Center for Biological Research, Spanish National Research Council (CSIC), Madrid, Spain
Patricia Boya, Department of Neurosciences and Movement Science, Faculty of Science and Medicine, University of Fribourg, Fribourg, CH-1700, Fribourg, Switzerland
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