Tsui-Chin Huang —BIO-PROTOCOL

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Pan-Chyr Yang Institute of Biomedical Sciences, Academia Sinica, Taiwan
3 protocols

Hsueh-Fen Juan Department of Life Science, Institute of Molecular and Cellular Biology, National Taiwan University, Taiwan
2 protocols

Yi-Ching Wang Department of Pharmacology, National Cheng Kung University, Taiwan
2 protocols

Hsuan-Cheng Huang Institute of Biomedical Informatics, National Yang-Ming University, Taiwan
2 protocols

Tsui-Chin Huang

Institute of Molecular and Cellular Biology, National Taiwan University, Taiwan

2 Author merit
Reviewer merit
Editor merit

2 Protocols published

Flow Cytometric Detection of Reactive Oxygen Species

Authors: Hsin-Yi Chang, Hsuan-Cheng Huang, Tsui-Chin Huang, Pan-Chyr Yang, Yi-Ching Wang and Hsueh-Fen Juan, date: 04/20/2013, view: 26338, Q&A: 2

Reactive oxygen species (ROS) are molecules containing hydroxyl radicals or peroxides with unpaired electrons. In healthy aerobic cells, ROS are produced naturally as a byproduct of oxidative phosphorylation, oxidoreductase enzymes, or metal ...

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Flow Cytometric Detection of Mitochondrial Membrane Potential

Authors: Hsin-Yi Chang, Hsuan-Cheng Huang, Tsui-Chin Huang, Pan-Chyr Yang, Yi-Ching Wang and Hsueh-Fen Juan, date: 04/20/2013, view: 17230, Q&A: 1

Mitochondrial membrane potential (Δψm) is an important parameter of mitochondrial function and an indicator of cell health. Depletion of Δψm suggests the loss of mitochondrial membrane integrity reflecting the initiation of the proapoptotic signal. ...

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Rodrigo Hoyos

Boston Children's Hospital

Hi, I've been using this protocol in fibroblasts, but even thou the staining works I don't get a depletion of the mitochondrial membrane potential in the cells fixed with fresh 4% paraformaldehyde. If I compare the MFI of both the fixed and non fixed cells, the MFI is higher in the fixed ones, do you have any suggestions on how to solve this? Since the fixation did not work properly I analyzed the membrane potential comparing live and dead cells without fixation, but I'm not sure that's a proper control for the loss of membrane potential. I will be thankful for any help you could give me.

many thanks

Rodrigo

12/2/2015 2:09:36 PM Reply

Hsin-Yi Chang

Kyoto University

Hi Rodrigo,

Positive charged fluorescent dyes such as DiOC6 accumulates in mitochondria due to their large negative membrane potential. Therefore, the dye can not be retained in mitochondria after incubation of any reagent causes membrane permeabilization, e.g. aldehyde fixation or detergent permeabilization. Alternatively, you can treat cells with mitochondrial uncoupling agents for mitochondrial depolarization. We've routinely used the paraformaldehyde as the positive control for membrane potential depletion and works well. You can check whether you washed the cells thoroughly after paraformaldehyde treatment and DiOC6 staining, and used the same cell number for staining.

Hsin-Yi

2015-12-02 17:23:34

MARIA MASOURA

UNIVERSITY OF BIRMINHAM

Rodrigo Hoyos

Boston Children's Hospital

Hsin-Yi Chang

Kyoto University