Hi Antoine, You need to complete those measurements on live mitochondria within 2 hours of collection, before the organelles start to lose their respiratory capacity and die. A colleague actually tired measure respiratory capacity on frozen mitochondria and got measurements of oxygen consumption but it would be inappropriate to assume that oxygen use was from fully functional respiring mitochondria. So don't be fooled if you try it.  As you know, you can still get measures of relative number of complexes (we do cytochrome oxidase), number of mitochondria, oxidative damage, antioxidant levels, etc. But once you freeze a tissue, unfortunately its dead.  I'm with you - I wish we could use frozen tissue. There are so many great questions we could ask it if were more convenient to measure RCR and complex activity.  Cheers, Wendy

In vitro measurements of individual respiratory chain complexes I-IV can be performed using frozen samples according to the attached publication which is, by the way, an excellent resource for assaying these complexes.

Cheers, Christian

Thank you very much for your answers, and indeed the paper in Nature protocol is my reference to assay such enzymatic activities. However, the samples are usually kept at -80°C and I don't  have access to a -80°C for this particular study, so I was wondering if there is a way to preserve the activity of ETC complexes at -20°C.

Hello Antoine, for how long would you need to store your samples at -20?

Thanks for pointing out that paper Christian, our lab is going to give these methods a try too!  

Antoine, please check this article: http://www.karger.de/Article/FullText/374012

I am wondering if it is not better if you store your blood samples refrigerated but not frozen...

Cheers, Christian

You are welcome, Wendy. The article is a very good collection of information on how to measure respiratory enzyme activity. Good luck with your experiments!

Hi All, I just came across this link.

Did anyone try the protocols for complex activities on frozen tissues and get it working?

I would like to give it a try (on frozen mouse tissue) if others have got it working nicely.

Cheers,

Mike

I did not have the time to try yet even if the question was posted two years ago; but in mammalian tissues flash-frozen and kept at -80°C it should be feasible according to the litterature. Good luck with those measurements!

Antoine

Hi Mike

We've had good success measuring complex activity on frozen samples, although we isolate mitos first and then store -80. We used the methods described by Spinazzi, Marco, et al. "Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells." Nature protocols 7.6 (2012): 1235.  Apparently, the method works well for whole tissue for all complexes, except complex II, which must be done on isolated mitochondria.  I hope that helps.

Wendy