A flow-cytometry-based protocol for detection of mitochondrial ROS production under hypoxia

Yun Yang; Guimin Zhang; Tao Yang; Jia Gan; Lin Xu; Hanshuo Yang

doi:10.1016/j.xpro.2021.100466

STAR Protocols (Jun 2021)

A flow-cytometry-based protocol for detection of mitochondrial ROS production under hypoxia

Yun Yang,
Guimin Zhang,
Tao Yang,
Jia Gan,
Lin Xu,
Hanshuo Yang

Affiliations

Yun Yang: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, NO.17, South of Renmin Road, Chengdu 610041, China
Guimin Zhang: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, NO.17, South of Renmin Road, Chengdu 610041, China
Tao Yang: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, NO.17, South of Renmin Road, Chengdu 610041, China
Jia Gan: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, NO.17, South of Renmin Road, Chengdu 610041, China
Lin Xu: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, NO.17, South of Renmin Road, Chengdu 610041, China
Hanshuo Yang: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center of Biotherapy, NO.17, South of Renmin Road, Chengdu 610041, China; Experimental and Research Animal Institute, Sichuan University, Chengdu, China; Corresponding author

DOI: https://doi.org/10.1016/j.xpro.2021.100466
Journal volume & issue: Vol. 2, no. 2
p. 100466

Abstract

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Summary: Hypoxia is known to stimulate mitochondrial reactive oxygen species (mROS) in cells. Here, we present a detailed protocol to detect mROS using MitoSOX staining in live cells under normoxia and hypoxia. Flow cytometry allows sensitive and reliable quantification of mROS by FlowJo software. We optimized several aspects of the procedure including hypoxic treatment, working concentrations of the staining buffer, and quantitative analyses. Here, we use HepG2 cells, but the protocol can be applied to other cell lines.For complete details on the use and execution of this protocol, please refer to Yang et al. (2020).

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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