I want to know the different techniques used in the quantification of mitochondria fusion and fission in an A549 cell line or other cancer cell lines.
Please help i am a Ph.D student researching on the quantification of mitochondria fusion and fission.
Thanks
This flowcytometry method may help you..
Detection and Quantification of Mitochondrial Fusion Using Imaging Flow Cytometry.
Nascimento A, et al. Curr Protoc Cytom. 2017.
Hi Momoh
Just saw your question, Don't know if it is too late now. Anyways, there are a couple of ways looking for fission/fusion dynamics of mitochondria.
1. You need to study the protein expression of some key regulatory proteins-
a) For fission just do western bloting for DRP 1 and its active form- pDRP s616
b) For fusion western bloting of Mfn1 and Mfn2.
Though there are other fission/fusion mediating proteins, western bloting quantification of these proteins would suffice your aim.
2. To prove your western blot data, you must do fluorescence imaging using Mitotracker Red CMX ros dye (thermo scientific), using confocal fluorescence microscopy. This dye stains live mitochondria and you can stain your cells grown on glass cover slips and after co staining the cells with DAPI (nuclear stain) you can visualise red mitochondria surrounding blue nuclei.
I am attaching some relevant publications for your further reference-
1. Article Mitochondrial fusion exploits a therapeutic vulnerability of...
2. Article Erk2 Phosphorylation of Drp1 Promotes Mitochondrial Fission ...
Hope this works for you...
Good luck
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