Measurement of intracellular and mitochondrial ROS levels

YL Yuan Liu
JG Ji-Zheng Guo
YL Ying Liu
KW Kui Wang
WD Wencheng Ding
HW Hui Wang
XL Xiang Liu
SZ Shengtao Zhou
XL Xiao-Chen Lu
HY Hong-Bin Yang
CX Chenyue Xu
WG Wei Gao
LZ Li Zhou
YW Yi-Ping Wang
WH Weiguo Hu
YW Yuquan Wei
CH Canhua Huang
QL Qun-Ying Lei
request Request a Protocol
ask Ask a question
Favorite

ROS and mitochondrial superoxide production were determined using a fluorescent dye chloromethyl-2′,7′-dichlorofluorescein diacetate (H2DCF-DA, Sigma, 35845) and a specific mitochondrial superoxide indicator MitoSOX Red (Thermo Fisher, M36008), respectively. Briefly, cells with specified treatments were washed with PBS and incubated with 5 μM H2DCF-DA (or 10 μM MitoSOX) at 37 °C for 30 min to load the fluorescent dye. Afterward, cells were washed twice with PBS, and trypsinized, centrifuged, resuspended in 500 μl PBS. Cells were kept in the dark until analysis by flow cytometry (Accuri C6, BD Biosciences).

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A