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Article

A Novel FACS-Based Workflow for Simultaneous Assessment of RedOx Status, Cellular Phenotype, and Mitochondrial Genome Stability

1
Comprehensive Alcohol Research Center, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA
2
Department of Physiology, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA
3
Department of Medicine, Section of Pulmonary and Critical Care Medicine, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Buyong Ma
Received: 3 January 2021 / Revised: 25 March 2021 / Accepted: 26 March 2021 / Published: 2 April 2021
(This article belongs to the Special Issue Computational Analysis of Proteomes and Genomes)
Intracellular reduction-oxidation (RedOx) status mediates a myriad of critical biological processes. Importantly, RedOx status regulates the differentiation of hematopoietic stem and progenitor cells (HSPCs), mesenchymal stromal cells (MSCs) and maturation of CD8+ T Lymphocytes. In most cells, mitochondria are the greatest contributors of intracellular reactive oxygen species (ROS). Excess ROS leads to mitochondrial DNA (mtDNA) damage and protein depletion. We have developed a fluorescence-activated cell sorting (FACS)-based protocol to simultaneously analyze RedOx status and mtDNA integrity. This simultaneous analysis includes measurements of ROS (reduced glutathione (GSH)), ATP5H (nuclear encoded protein), MTCO1 (mitochondrial DNA encoded protein), and cell surface markers to allow discrimination of different cell populations. Using the ratio of MTCO1 to ATP5H median fluorescence intensity (MFI), we can gain an understanding of mtDNA genomic stability, since MTCO1 levels are decreased when mtDNA becomes significantly damaged. Furthermore, this workflow can be optimized for sorting cells, using any of the above parameters, allowing for downstream quantification of mtDNA genome copies/nucleus by quantitative PCR (qPCR). This unique methodology can be used to enhance analyses of the impacts of pharmacological interventions, as well as physiological and pathophysiological processes on RedOx status along with mitochondrial dynamics in most cell types. View Full-Text
Keywords: FACS; RedOx status; mitochondria; mitohormesis; mtDNA FACS; RedOx status; mitochondria; mitohormesis; mtDNA
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MDPI and ACS Style

McTernan, P.M.; Katz, P.S.; Porretta, C.; Welsh, D.A.; Siggins, R.W. A Novel FACS-Based Workflow for Simultaneous Assessment of RedOx Status, Cellular Phenotype, and Mitochondrial Genome Stability. BioChem 2021, 1, 1-18. https://0-doi-org.brum.beds.ac.uk/10.3390/biochem1010001

AMA Style

McTernan PM, Katz PS, Porretta C, Welsh DA, Siggins RW. A Novel FACS-Based Workflow for Simultaneous Assessment of RedOx Status, Cellular Phenotype, and Mitochondrial Genome Stability. BioChem. 2021; 1(1):1-18. https://0-doi-org.brum.beds.ac.uk/10.3390/biochem1010001

Chicago/Turabian Style

McTernan, Patrick M., Paige S. Katz, Constance Porretta, David A. Welsh, and Robert W. Siggins 2021. "A Novel FACS-Based Workflow for Simultaneous Assessment of RedOx Status, Cellular Phenotype, and Mitochondrial Genome Stability" BioChem 1, no. 1: 1-18. https://0-doi-org.brum.beds.ac.uk/10.3390/biochem1010001

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