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

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

STAR Protoc. 2021 Apr 20;2(2):100466. doi: 10.1016/j.xpro.2021.100466. eCollection 2021 Jun 18.

Authors

Yun Yang¹, Guimin Zhang¹, Tao Yang¹, Jia Gan¹, Lin Xu¹, Hanshuo Yang^{1

2}

Affiliations

¹ 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.
² Experimental and Research Animal Institute, Sichuan University, Chengdu, China.

Abstract

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).

Keywords: Cell Biology; Cell-based Assays; Flow Cytometry/Mass Cytometry; Molecular/Chemical Probes.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Flow Cytometry*
Hep G2 Cells
Humans
Mitochondria / metabolism*
Phenanthridines / chemistry*
Reactive Oxygen Species / metabolism*

Substances

5-(6'-triphenylphosphoniumhexyl)-5,6-dihydro-6-phenyl-3,8-phenanthridinediammine
Phenanthridines
Reactive Oxygen Species