Hi Shilpa,

Mitochondrias are the most dynamic organelles and its really difficult to find out its exact number per cell. Even if you lyse the cells you will not able to come up with exact number of mitochondria per cells, that would be tentative as mintochindrias continue to burst and fuse.  Acc . to me you can find out different fractionations of the cells like , membrane, cytosolic, mitochondrial and with the help of immunoblotting you can come up with mass ratio for the representation.

 Other methods what I  read are:

1- qPCR to normalise a mitochondrial DNA amplicon to nDNA

2-Confocal microscopy to determine mitochondrial mass by dividing TMRM fluorescence to total cell fluorescence (used calcein blue for total cell mass).

But it  would be the representative data.

You can see this link also:  http://www.ncbi.nlm.nih.gov/pubmed/317012

All the best

Abhinav

Thanks Abhinav. Well i have read about stain area optimisation for mitochondrial numbers some time back but did not get the way to calculate. qPCR of course is a way to do it.

Hi Shilpa,

I agree with Abhinav. There is no way to count the exact number of mitochondria per cell. If you use fluorescent dyes to measure mitochondria TMRM is not the best choice.  There are dyes that specifically stain mitochondria independent of their membrane potential like MitoTracker or NOA. They allow you to measure differences in mitochondrial mass, for example after some treatment even when the treatment affects mitochondrial performance.

Best Nathalie

It's also worth considering what you mean by "mitochondrial number": as several posters have pointed out, mitochondria are not static (and in fact, the concept of "a mitochondrion" is more of a misnomer derived from E.M. artefacts). Under most normal conditions they form highly branched, but interconnected, reticular structures, so the answer would be "about...one, but it's a big one". If you stress cells however, the mitochondrial reticulum can fragment, so the same total volume, but now in tons of tiny sausages.

Mitochondria also have different properties, dependent on which cell they reside in.

Mitochondrial density/volume (as determined by fluorescence microscopy or similar) is a measure, but far from the only one, and it tells you nothing about the composition of those mitochondria. Are they highly oxidative and concerned with ATP generation? If so, from fat or from sugar? Or are they highly biosynthetic? Are they carefully arranged, cytoskeletally-anchored calcium sinks, or are they more freeform? And so on.

And of course, all these properties can change without any change in the apparent mitochondrial density, and conversely, mitochondrial density can change without any apparent change in these properties. 

So really you should work out exactly what question you want to ask. If you're concerned with some sort of absolute mitochondrial number, then yes: mito genome count is the way to go, since these are absolutely discrete, countable elements (though be aware that mitochondrial density and behaviour can change without any significant change in mitoGenome count). If you're concerned with oxidative capacity, then maybe westerns for ETC components or similar is the way to go. If you're interested in changes in mitochondrial network, then fluorescence microscopy is the way forward (though to be honest you should do this anyway, because it makes pretty pictures and these are always good for publications).

A final thought to consider: cells usually tend to fragment their mito network just prior to division. Partitioning into daughter cells will essentially follow binomial distribution, so the more individual mitochondrial elements they can make, the better the chance of getting an equal number in each daughter cell. So if you're really interested in some sort of number and you're good at spotting cells getting ready to divide, then using some sort of vital dye, a fluorescence/confocal microscope and a lot of patience might be workable. Wouldn't recommend it, though. 

I think you should also take into account that qPCR will tell you how many copy of mtDNA are per cell, but not how many mitochondria per cell, and this is because each mitochondrion contain multiple mtDNA copies... You can also purify mitochondria from the hole extract, and then mesure proteins concentration in mitochondria fraction and total extract (e.g. BCA method), this will give you an estimative ratio of mitochondria per cell, but is not very accurate either (this will never give you the exact nomber). I totally agree with the comments above, there is not a unique number of mitochondria because is a dynamic network in contunous fussion/fission.

Mitotrackr Red is a lipophilic cation and accumulates into mitochondria due to the mitochondrial membrane potential; its labeling is not at all independent of the membrane potential as can be seen by titration with classic uncouplers like CCCP.

Gilmore and Wilson did a nice study back in 1999 (Cytometry 36:355–358 (1999))

They state:-

"In summary, our results are consistent with CMX-Ros [Mitotracker red] being a valid probe for ΔΨ in intact cells but only when the cells are stained and analyzed immediately. Fixation of CMX-Ros [Mitotracker red] stained cells apparently preserves some association of CMX-Ros with mitochondria but the extent of this association cannot be taken as a true indication of ΔΨ in the cells prior to fixation"

Personally, fixing cells in 4% ice-cold PFA, washing with cold PBS and then measuing signal within 6 hours work very well

http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0320(19990801)36:4%3C355::AID-CYTO11%3E3.0.CO;2-9/pdf

Thanks everyone. Great suggestions.!

mtDNA analysis appears to be a "popular" technique, but I'd be cautious of its interpretation. I had every indication that the mito # in my treatment group was high, based on qPCR of genes from the TCA cycle, respiratory complexes and their activities measured using simple enzyme assays and high resolution respirometry (Oroboros); still the mtDNA qPCR reported no difference in my two experimental groups. It took TEM imaging to finally reveal that the mitogenesis had indeed occurred. TEM is considered the gold standard for mito work, besides it can show any ultrastructural changes if any.

People, you will understand mitochondria much better, if you will stop treating them as "a very dynamic organelles". Mitochondria are THE ESSENCE OF LIFE, not just another, though dynamic, organelle. And  because a cell might require different amount of energy, their number may go up or down. They ARE dynamic, but just counting them make little sense. Particularly relying on counting mtDNA. Dear Shilpa, what exactly do you want to understand?

Depending on the type of a cell, the number of mitos cam vary form just few (1-2) in lymphocytes to several thousands (hepatocytes). In some tissues mitochondria are very large and branched, Under microscope you might thing there are 10 different mitochondria, but it is just one (heart cell). As you see, your question is not as simple as it might look, but also is not complete and wise. I would suggest that you would know more about mitochondria.

Thanks Alexander. I too believe mitochondria are the essence of life and therefore a challenge to understand. Their behaviour under different conditions are complex to understand. I have done lot of TEM in TB infected cells and seen the morphological differences between the groups. But since TEM is not very much easy to access all the time so now relying on other techniques of Confocal microscopy. Though have done Mitotracker, but this resolution is not enough to interpret.  What I am looking at is asking the question First - as to quantify the alteration at the level of mitochondria in a treated versus untreated cell. Now if I want to see reversal, would the  treatment reverse the alteration? How should i go about this ordeal? 

Can you answer the following questions:

1. Do you use antibiotics (PenStrep)?

2. What medium you use, and what concentration of glucose you have in the medium?

3. Do you have ssupplements in the medium?

4. What is the origin of the cells?

Thank you.

Answer to your questions are as under-

1. Yes RPMI 1640 includes PenStrep.

2. RPMI 1640. Has about 2mg/ml glucose 11.1mM concentration

3. Yes these ready mix medias include supplements 

4. Monocytes but for experiments differentiated to macrophages using PMA

Thank you