I have been trying to isolate intact mitochondria for an uptake study.
Currently I harvest cultured cells using trypsin, ressuspend cells in isotonic sucrose buffer (Sucrose 0.25M, EDTA 1mM, HEPES 10mM pH 7.4 with freshly added protease inhibitor cocktail), lyse cells using a Dounce homogenizer and use the centrifugation method in order to isolate my mitochondrial fraction.
Once I have my isolated fraction I ressuspend it in the same sucrose buffer previously described and am trying to determine if the isolated mitochondria has intact membrane or not.
I have been trying to use the "Isolated Mitochondria Staining Kit" from Sigma Aldrich that uses JC-1. From my understanding JC-1 monomers have green fluorescence and once accumulated into the mitochondria (due to it's transmembrane potential) it would form aggregates that shift to red fluorescence.
I was comparing results of mitochondrial fractions with and without treatment using valinomycin (which should dissipate mitochondria membrane potential) and obtained the same red fluorescence value for both.
I have also measured the green fluorescence of 1) Water 2) JC-1 solution 3) JC-1 solution + mitochondria fraction and 4) JC-1 solution + mitochondria fraction treated with valinomycin. Interestingly I got higher green fluorescence values for 3 and 4 and got much lower value for JC-1 solution, which didn't differ at all from water.
Shouldn't I see high green fluorescence for the JC-1 solution since it would have plenty monomers of the molecule?
I wold appreciate if anyone with more experience in this area / type of assay could help me.
Hello Leticia,
JC-1 is tricky to use. For some it seems to work well enough, others report problems with realiability (and a tendency to aggregate in the absence of a mitochondrial membrane potential).
You can find an interesting discussion on this topic here:
"Mitochondrial Biology: New Perspectives: Novartis Foundation Symposium 287",
edited by Derek J. Chadwick and Jamie Goode (2007).
I would suggest to switch to another dye, like TMRM or TMRE. Here you can find an interesting comparison of several dyes that are frequently used to measure the mitochondrial membrane potential:
Article Mitochondrial membrane potential probes and the proton gradi...
I hope that this might be useful to you.
Best regards,
Christian
Dr. Scheckhuber,
Thank you for taking some time to answer my question.
The files you attached and recommended are very helpful and I will try some other dyes as you suggested.
Best regards,
Letícia
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