Normally, I use MTT assay which detect mitochondrial activity directly and you can measure them using microplate reader at OD540 or 570. That's work!!

Thank you for your answer! I am not sure if I could use this type of assay with the tissue homogenate which does not adhere to the microplate.

If you want to study the functional state of mitochondria in your homogenate you should use Mitochondrial Membrane Potential Assay Kits (try to find on Internet). There is marker enzyme for mitochondria (succinate dehydrogenase). I am not sure if it will be helpful for you. Works usually , when you need to be sure in isolated pallet that you successfully isolated mitochondria

Hi Maciej

I agree with Malgorzata. You may check this link or similars to determine COX and SDH activities. I am not sure which one is dominant in myocardium, but they may be used histochemically and (follow the link) spectrophotometrically

http://books.google.es/books?id=RO2_zQaVltwC&lpg=PT316&ots=zlNykL6p2H&dq=COX%20SDH%20activity&hl=ca&pg=PT315#v=onepage&q=COX%20SDH%20activity&f=false

Hope that it will help you!

Best of lucks!!

Manuel

also check mitochondrial membrane depolarization (i.e, change in membrane potential) with JC-1 fluorescent dye in FACS.

These assays are all highly appropriate. Additional I would examine mtDNA accumulation.

George Perry

Hey Maciej, whats about using a simple ATP determination kit? Available at invitrogen you just have to have an luminiscence reader. Have a look on it here

http://products.invitrogen.com/ivgn/product/A22066?ICID==%3Dsearch-product

I don't know how detailed you have to examine mitochondrial function or activity. Dependent on the pathomechanism you're interested in there a specificly affected proteins which show for instance changes in phosphorylation pattern (pStat3 S727, caspases a.s.o.). Of coarse I agree with the last two comments. However, staining mitochondria with ratiometric dye JC-1 with subsequent FACS sorting is the easiest way when a FACS is available.

Best Nicolas

I do not work with this, but a friend of mine measures mitochondrial activity in insect extracts, she might be able to help.

Follows a link to one of her publications. I would advise having a look to see if methods meet your demands, and then try to contact her (first author).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773413/

Hope it helps.

I'm not sure about the functional state or the activity level of mitochondria in cardiomyocytes are the same thing. You may try with SDH but this only informs about the enzyme activity, not mitochondria functional state. The very classics way is a direct measure of oxygen consumed (Clark electrode) by a mitochondrial preparation (by subcellular fraccionation) in the presence of a defined quantity of ADP Vs the same in absence of ADP (State 3/State 4 value). Nevertheless, please take a look on papers by Dr. Orian Shirihai for methods with fluorescent dyes. (but some caution and expertise is needed) Good luck

Completely agree with Eduardo....You can use Oxygen Biosensor plates (BD Biosciences) to measure O2 consumption if you do not have enough tissue or a clark electrode. This analysis should be analyzed in light of enzyme activities, and very important mitochondrial membrane potential using JC1 or TMRM. Look at Samudio I, et al. JCI 2010; and Samudio I, et al. Cancer Research 2008 for some helpful techniques.

Good luck!

Hi! In fact, I am also interested in this field. Recently a very good article concerning your problem has appeared:

http://www.biochemj.org/bj/435/0297/4350297.pdf

If you want to carry out a comprehensive study of mitochondrial processes, i truly recommend you to read this article.

Best wishes, Manko Bohdan

Thank you for the clues and advice. You all were very helpful as this is my first approach to the mitochondria topic. I'll do the reading now :)

Sorry for been a bit late)

I mostly agree with Eduardo Kessi: oxygen consumption (polarography, Clark electrode) and ATP generation (luciferase) are main direct ways to understand functionality of mitochondria; both can be determined using plate-reader. All the others (mitochondrial potential, SDH, etc.) are indirect and suit better to investigate where mitochondrial defect is.

If you are eager to use potential sensors, I recommend TMRE/TMRM and Rhodamine123 (at working concentrations 50-150 nM for 500k cells they can be used for quantitative measurement. To use them for FACS, Rho123 is very like FITC, forTMRE/TMRM you need either green or yellow laser, or powerful 488 laser like ones, used in FACS Aria). Second, to normalize mitochondrial potential you need a potential-independent mitochondria-specific stain, preferably one of MitoTracker family. MitoTrackers Red and DeepRed were shown to be independent of mitochondrial potential, since they got inside of mitochondria. NAO is not recommended for its nonspecific staining. So, you get relative potential per mitochondria volume unit, that usually correlates with mitochondria function. To summarize, appropriate FACS panel seems like this: AnnexinV, TMRM, MitoTrackerDR + cardiomiocyte markers (CDs, etc). At least 2 lasers are needed.

I don't recommend JC, DiOC ans Di-S-C, and formazan (MTT,MTS) families to stain mitochondria. JC family cannot be used for quantitative measurements and better not to be used for yes-no measurements since there are many factors, except potential, that influence on dye accumulation. If you are working with cells showing not very high poteniial, just don't use it. The others show low specificity, toxicity and, mostly, autoquenching.

Yours sincerely, Sergey

Thank you Sergey for your comprehensive answer. Did you took into account that I have to do the asessment in the deep-frozen tissue sections? Is it possible to adapt these FACS-based methods for tissue. Cells isolation or smth?

Sorry for a late answer. FACS-methods can be fully used only on cell suspensions, if you want, you can try to homogenate your sections and run it on a low speed, but I wouldn't recommend you to do so. To stain mitochondria after cell fixation (or other killing method) without mitochondria-specific antibodies, you can try NAO since it is reported to bind to cardiolipin, but I really don't know how specific and strong the signal will be. I tried the method just once, to make sure it works.

It always hard to say about fixed or frozen cells how functionally active were they when they were alive. Using only biochemical methods you can't be completely sure, you need to verify yourself with Clark electrode on living cells.

as i read papers using TMRE alone show qualitative images as well as calculation of Fluo intensity by microscopy software. how exactly the membrane potential is calculated in a formula? is this in need of other equipments than microscopy or knowing the intensity is enough? I appreciate if anybody share a good ref with me