Improve Research Reproducibility A Bio-protocol resource
- Home
- Protocols
-
Hi, I've been using this protocol in fibroblasts, but even thou...
RH
Rodrigo Hoyos Dec 2, 2015
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 o Read more 
2
Views
Answer
Recommend
Follow
Share
1
answer
Sort by: most helpful / most recent
Hsin-Yi Chang Author Answered Dec 2, 2015
National Defense Medical Center
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
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
0 helpful
0 unhelpful
0 comments ShareMy answer
Write your answer...
References (optional)
Add more
Post an Answer