Please check the following references:

https://www.ncbi.nlm.nih.gov/pubmed/17406588 and

https://www.nature.com/articles/nprot.2012.058

I am positive that these articles can guide you well.

Regards, Christian

For the viability O2 consumption (polarography) is the answer but probably is too cumbersome if you want an answer in the few hours you have between isolation and transplantation. Transmembrane potentials will give you a correlated value but with less precision.

For the purity question there is no easy answer, because all protocols produce mixes. Typical this is assessed by western blot, but it is out of question in your time line.

Maybe you can solve both problems by facsing the mitochondria stained with JC1. I did this in past and works well, but I was not trying to recover mitochondria at the end. I don't think you can wash out JC1, so if you want to do the same, you'll need another type of sensor, like a recombinant protein.

Measure respiration with various substrates and substrate mixtures and compare with the selected control. For control the best are brain and heart mitochondria, Liver is not a suitable control because in many ways liver mitochondria are unique.

Monitoring respiration with multiple substrates eg. flutamate, succinate, ascorbate+ TMPD is advisable. For purity check you can asses succinate dehydrogenase activity using PMS+ DCIP system. For gettng beter perpective consult Method in Enzymology Vol X.

I have to note that measuring the activity of cytochrome oxidase with ascorbate + TMPD has little sense. One of the reasons is that the method itself has many hidden complications, which very often make the results useless. The other reason is that Complex IV is in a large redundancy. Succinate + rotenone as a substrate is absolute nonsense. by itself succinate better to use in combination with glutamate or pyruvate or palmitoyl-carnitine. The details of succinate usage as a substrate I have published recently in Analyt Biochem. (see my Res.Gate page).

You have miss red my suggestions. I have said measure respiration with ascorbate+TMPD and not cytochrome oxidase activity. For the latter you use reduced cytochrome c as the substrate. While I suggested measuring respiration with glutamate and SUCCINATE I also suggested minitoring succinate dehydrogenase activity with PMS+ DCIP.

Dear Surendra,

To study cytochrome c oxidase (Complex IV) , reduced cytochrome c is used when work with submitochondrial particles. Ascorbate + TMPD is used with the whole mitochondria.