what do you mean by "I cannot manage it"?

I am using the parr-bomb system and the OMM is perfectly intact.

In addition you can use the siringe (27G) to destroy the cell membrane and perform an easy 800g + 8000g enrichment steps.

I mean that I cann't isolate intact inner membrane. It is complutly destroyed.

try with the 2 suggestion I gave you. I am working with intact mitochondria for physiology studies...

Check out this article:

Antonia Martínez-Morúa , María G. Soto-Urquieta , Elena Franco-Robles , Ismael Zúñiga-

Trujillo , Alejandra Campos-Cervantes , Victoriano Pérez-Vázquez & Joel Ramírez-Emiliano (2013): Curcumin

decreases oxidative stress in mitochondria isolated from liver and kidneys of high-fat diet-induced obese

mice, Journal of Asian Natural Products Research, DOI:10.1080/10286020.2013.802687

The isolation of mitochondria from cells is easy enough. The main problems consists on the very low yield, thus it would be better, if possible, to use cell homogenates with control made of the mitochondria intactness. Since the isolation procedure could differ in dependance of the cells, it would be usefull to know what kind of cells you are dealing with.

If the IM is destroyed, maybe you have a problem with a buffer ?

I would avoid any proteases since they really damage the mitochondrial function

We use a siringe (27g size) using HeLa or MEF cells. This works fine. You can test intact mitos by blotting for cytochrome c. If your Tim protein is digested with proteinase K this implies that your outer mitochondrial membrane is already broken. Therefore cytc will also leave mitochondria. You should really check your buffer system. And keep everything on ice! Did you prepare and test fresh mitochondria? Just asking because mitochondria from mammals dont like that( instead of yeast mitos).

Sorry. I wanted to say that you should not freeze mitochondria and then thaw if you want intact mitos...

I would not recommend using the syringe, as it is a stress for the mitochondria and it won't give you consistent results. The only trick to healthy mitochondria when using the Potter homogenizer is to carefully setup the right speed and the right amount of times you go up and down, depending on your cell type. That will give you a cell homogenate. A centrifugation at 1,000xg, 5 min at 4 degrees will help get rid of nuclei and cell debris. To recover more mitochondria I use the Potter homogenizer twice (I homogenize again the pellet from this centrifugation and add the supernatant from this centrifugation to the previous one) Another centrifugation at 12,000xg, 10 min at 4 degrees and you have your isolated mitochondria in the pellet. Wash a couple of times and you are ready to go. Ice cold MSHE buffer, pH 7.4 for every step (the buffer you are already using) works very well. I get healthy mitochondria every single time. Good luck!

Please, check this article published at Nature Protocols. It is working very well!

PMID: 17406588

Frezza C, Cipolat S, Scorrano L.

Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.

Nat Protoc. 2007;2(2):287-95.

If isolating mitochondria from cells fails, or gives you too low a yield/viability, you could try and study the mitochondria in situ in cells that have had their plasma membrane disrupted. These mitochondria are far superior to the isolated ones in terms of function and normal connections with the ER and cytoskeleton. The plasma membrane can be permeabilised with very low concentrations of digitonin on ice, for a fixed timed period. Find out the optimal concentration/time measuring the release of a cytosolic marker enzyme e.g. LDH. At the minimal digitonin required to release most of the releasable LDH (about 60%) the mitochondria remain intact, if the permeabilised cells are washed and re-sedimented after their timed incubation with the digitonin.

In cells that are a little bit harder, I suggest sonication. I do syringing, plus homogenizer, plus sonication. This has given me the highest yield. I use both cyclophilin d and coxiv antibodies to test mitochondrial presence. Hope this helps!

I sugested the article of Frezza et al. 2007 of Nature Protocols vol 2 n2, that have good indications.

I agree with Carlos and Dongryeol -- The protocol from Frezza et al works very well.

Point of clarification: The Frezza et al protocol works wonderfully for intact and functional mitochondria. If you need highly pure intact mitochondria but don't need them to be functional, then adding an ultracentrifugation step using a percol or ficoll gradient will be necessary.

I also use the Frezza et al. Nature Protocol. Bonus: at the end of the protocol, they tell you how to test for mitochondrial function, so you can see whether the mitochondrial membranes are intact.

We isolate mitochondria from HeLa cells and yield is quite high. We've optimized to use 1.5 mL Eppendorf tubes on a table-top centrifuge, so it's a really easy protocol, takes about 1.5 hr.

Results summary attached as a .JPG

For controls, we do Western blots with anti-COX IV (positive control, integral inner mitochondrial membrane protein), anti-calnexin (ER control), and anti-beta-tubulin (mostly cytoplasmic) (Panels B-C).

We have also tested for seepage of mitochondria (COX IV) out of each fraction in this protocol and see no COX IV in any other fractions and a bright COX IV band in the final mitochondrial enrichment (Panel A). Works great!

Highly recommend this protocol, especially if you're having trouble keeping membranes intact.

Here is our protocol we adopted form one article and expanded to other cell types. We checked the mitochondrial "health" by measuring oxygen consumption and/or mitochondrial membrane potential with TMRM or safranine O.

And here is a file itself)))

Dear Sofya,

we have isolated mitochondria, other organelles as well as submitochondrial fractions from rat liver (and skeletal muscle of rats) as described in K. Pamp et al. Biochimie. 2008 Oct;90(10):1591-601. Marker enzyme activities were used to verify the purity of the fractions. However, isolation protocols largely depend on the kind of tissue or cells used. I have attached the respective article for your information and hope that this will help you.

Best regards,

Frank

On students experimental lab we use for mitochondria isolation technique similar to that given by Maria Franco. We homogenize tissue (liver or heart tissue) in Potter teflon homogenizer in isotonic sucrose (A) samples were lysed in the buffer: 10 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1%Triton X-100, 2 mM MgCl2, 0.1 M ditiotreitiol and protease inhibitor cocktail, according to Blobel and Potter technique modification. After homogenates centrifugation at 10000 x g (10P fraction) in pellet you will have mitochondria. When pellet is contaminated with lipids the phase contained hypertonic sucrose (B) is needed and that rewash in isotonic sucrose A. In pallet we check mitochondria presence by detecting succinate dehydrogenase activity.

Always work. Good luck.

I would also support Frezza et al 2007 publication (Nat Protoc). It works really well!

You can also take a look at the attached paper where you'll find another methods.

Dear Sonya, teflon homogenizer is not a good method for the cells. From my account on ResearchGate, or directly from PLOS One you can load my paper on Prostate cancer where I describe the method which allows easily with good quality isolate mitochondria from all types of cells. I call this method "Hypothonic method".

However, there are so many more tricks and hardhisps to avoid. You can Find of Amazon.com for $25.00 (your lab will be not defolted) my book "Practical Mitochondriology" where I describe the many things I have got to know during my 45 years of experiments with mitochondria.

See "Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells"

Wieckowski MR, Giorgi C, Lebiedzinska M, Duszynski J, Pinton P.

Nat Protoc. 2009;4(11):1582-90. doi: 10.1038/nprot.2009.151. Epub 2009 Oct 8.

OK, colleagues. Since here people advised various methods and protocols, I have made some critical comparisons and commented briefly on them, but with the main focus on the cell mitochondria.

The method you select basically depends on what is your purpose for isolation of mitochondria. If you are interested only in mitochondrial DNA o4r proteins (Westerns, etc.), than you can use any method with any protocol. It does not matter much, you will get your mtDNA. But, if you plan to study the mitochondrial functions, than it is another matter. You have to get functioning mitochondria with good respiratory control and good rates of respiration. Therefore, my comments regards these mitochondria.

First comes the protocol advised here by Dr. Irina Perevoshchikova.

1). She, as well as in the papers by Fernandez-Vizarra et al. 2010, and Frezza et al. 2007, it is recommended to harvest cells at 85% or almost 100%. This is not good, because I myself (see my Paper in PLOS One) and other people observed that in cells harvested at confluence more than 80% or close to 100% confluence, the isolated mitochondria have the signs of initiated apoptosis. For prostate cancer cell mitochondria at 60% the membrane potential was higher than 200mV, at 80% - only 160 mV.

2) The usage of Potter-Elvejem homogenizer with tight plunger (pestle) damages mitochondria and the yield is not high (better to say, far from maximal).

3) Spinning the homogenate at 3000 rpm, which with SS-34 rotor gives about 1000 g (do not remember exactly). The same is also with the protocols by Fernandez-Vizarra et al. 2010, and Frezza et al. 2007. The point is as follows: in most cells, the mitochondria are huge circular structures often forming a net. Therefore at accelerations higher than 300 g, much mitochondria become sedimented with the cell’s debris. I know this from my experience. Therefore, you need much lower initial speed and a couple of additional steps to separate from the nuclei and other lighter debris and that is why purification hels.

Dr. Malgorzata Rogalinska advises the usage the isolation buffer, which contains 1% Triton X-110. Regardless of other things, this alone will render your mitochondria completely dysfunctional because of severely damages to the inner membrane. The method, evidently, is highly useful, if you are interested just in mtDNA or determination of proteins.

The method, which was highly recommended here by several colleagues, namely the method by Frezza et al. 2007, has several serious pitfalls for the isolation of functional mitochondria, which I briefly comment:

1) Recommendation to grow cells in the presence of streptomycin will lead to mitochondria without respiration (see my paper in PLOS One);

2) 100% confluence – see above (though with PenStrep it does not matter);

3) The authors also recommend the usage of EDTA. EDTA will chelate Mg, and this results in the opening the pore for protons and there will be no reverse electron flow and thus ROS production. Though, if you have in the incubation buffer 2-5 mM MgCl2 this will render again normal energization.

4) In general, the recommended incubation buffer is far from being optimal.

The protocols for isolation of mitochondria from organs by Pamp et al., Fernandez-Vizarra et al. 2010, Frezza et al. 2007 and Wieckowsski et al. 2009 are not completely fir for the isolation of the cell mitochondria. For each cell line you have to make some adjustments for optimization of the protocol.

In conclusion, did you noticed that in neither paper describing how to isolate cell’s mitochondria, nobody presented the actual polarographic trace for the cell mitochondria or presented the respiratory rates.

I have attached my PLOS One and other paper with the hypotonic method of isolation of mitochondria from prostate cancer cells and human lymphoblastoid cells. Fernandez-Vizarra et al. 2010, also used hypotonic treatment. My method was published in

Here is another paper. You cannot attach two files in the same message (may be I am wrong).

During my PhD I accumulated a lot of experience on the protease K (PK) treatment of mitochondria. One important point first: oxygen consumption as used in the Frezza et al protocol is a good assay for mitochondrial ACTIVITY but a bad marker for INTEGRITY (since mitochondria with a damaged inner membrane show an even higher respiration!).

Using protease K you can successively probe the integrity of the mitochondrial membranes. A fully intact outer membrane (OM) should protect all intermembrane, inner membrane and matrix proteins against digestion. However, you have to take into account that even during an optimal mitochondrial isolation at least 10% of the mitochondria have a damaged outer membrane! A damaged OM will become visible by a partial digestion of Tim23, since it exposes a domain on the intermembrane space side. Similarly, the OM preprotein channel Tom40 becomes digested when the OM is damaged. Please note that even with a damaged OM, mitochondria remain fully functional (at least concerning their metabolic activities)! All matrix proteins will be resistant against protease K as long as the inner membrane is not compromised. If you see PK-mediated digestion of matrix proteins, your mitochondria have been destroyed (any abundant matrix protein may serve as a suitable marker in Western blots).

Important: Take into account that some proteins are intrinsically resistant to PK digestion (independent of their accessibility inside mitochondria). This can be checked easily if you include a control experiment where you have destroyed the mitochondria on purpose by a treatment with 1% Triton X-100 before you do the digestion assay. In this case all proteins are accessible and should also be completely digested when you add the PK.

Good luck with your experiments!

We were also able to do this. Check out:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0014359

Dear Wolfgang Voos,

The problem is that all proteins after the PK treatment is degested, I checked it with TIM23. could you tell me what protease K you use? I think the problem could appear because the PK quality.

Thank you in advance,

Sofya.

Hi Sofya,

I try to avoid endorsing any specific lab supplier in public but if you type the following number in an internet search engine, you may get a link to the source of our PK: 03115879001

Please take into account that the digestion conditions are crucial!

For digestion with PK we use a final concentration of 100 ug/ml and incubate for 15 min on ice (!). Then the digestions need to be stopped by adding the protease inhibitor PMSF (final conc. 1 mM; stock solution 100 mM in ethanol). Mix quickly and thoroughly and incubate again 15 min on ice. Under these conditions, all mitochondrial proteins except those exposed on the outer face of the outer membrane should remain intact, if you have high quality mitochondria. If you still get full digestion, there is something wrong with your mitochondria preparation (as I mentioned already, a certain degree of digestion of Tim23 might not be avoided - better use a matrix protein as a marker to check for the integrity of the inner membrane).

Wolfgang

There are several ways. The one that preserves mitochondria integrity is described in this reference: Kook, S., Zhan X., Cleghorn, W.M., Benovic, J.L., Gurevich, V.V., Gurevich, E.V. Caspase-cleaved arrestin-2 and BID cooperatively facilitate cytochrome C release and cell death. Cell Death Differ 21 (1), 172-84 (2014). You can find more details in the original paper referenced in the one above.

I wish to get suggestions on any available protocols for mitochondria isolation by ultracentrifugation   

The method I have described in my book "Practical Mitochondriology" (see mt page in Research Gate, or paper version on the Amazom.com) is working with many types of cells, even those, which are resistant to detergents. It is based on hypotonic swelling of cells, which then blow up during strokes in the Dounce homogenizer.

Can someone suggest good mitochondrial marker proteins for Drosophila that would have (commercially) available antibodies? I've got an impression that there isn't much for endogenous fly proteins. (Though, it's always possible to use transgenic flies which have e.g. GFP in mitochondria.)