Hi Eugenio

The best system to study all of the parameters you mentioned, except DNA damage, is the Oroboros O2K Oxygraph system (http://www.oroboros.at/index.php?id=oxygraph) with the appropriate set of modules and accessories. We have extensive experience in studying mitochondria function and ROS production using state-of-the-art facilities in the Center for Aging and associated Diseases in Zewail City of Science and Technology, Egypt (http://www.zewailcity.edu.eg/research-institutes/hims/caad/) . We are willing to collaborate on such a project. Please let me know ([email protected]).

Thanks,

Why the Oroboros system is the best? Any references to that?

There are many of competing technologies/companies proposing tools to study the mitochondrial (dys)function by analysis of OxPhos, extracellular acidication and ATP. These include Seahorse and Luxcel Biosciences. In any case you will need some equipment, at least standard plate reader plus some dyes.

You can check these works:

http://www.ncbi.nlm.nih.gov/pubmed/21726199

http://www.ncbi.nlm.nih.gov/pubmed/22005712

We've used Seahorse Technology with some collaborators in Liverpool, UK, as well as mt and ROS dyes along with flow cytometry and confocal imaging in our labs. Dyes can be tricky (both using them and interpretation - many variations in protocol and controversy regarding specificity) but great results with theSeahorse analyzer http://www.seahorsebio.com/ - a big expense though.

Dear Eugenio

It is always best to keep things simple and to get help from those working in the field. The easiest way to decide whether a drug alters mitochondrial function is to ask whether oxygen consumption changes. This is straightforward to measure with relatively simple, inexpensive systems, and you can ask whether oxygen consumption changes at rest, whether the maximal capacity is altered and whether the consumption attributable to ATP synthase activity changes. I see that you are from Santiago de Compostela - there are groups there working on mitochondria and it would be wise for you to talk with them (I am aware of the groups of Soto-Otero and Mendez-Alvarez). If you are familiar with microscopy and have easy access to fluorescence imaging systems then you can ask - again very easily and inexpensively - whether mitochondrial membrane potential or redox state are affected by your drugs. There is a huge literature on all this - it is not too difficult.

My best wishes

Michael Duchen

Hi Eugenio,

Of course there are alternatives to measure the parameters you are interested in. For example you can measure the activities of respiratory complexes, using spectrophotometric assays ( with self-made protocols or even with commercial kits from mitosciences (now abcam)). You can also measure the ATP production or content, ROS production in the same way using specific dyes ( for ROS production is also possible to do flux cytometry or analysis of SOD expression by RT-qPCT for example). As some people commented, Seahorse or Oroboros equipments allow you to measure the oxygen consumption, acidification...etc. For measuring mtDNA damage or mt-protein expression you can use RT-qPCR or Western blotting. I enclose some publications from my PhD where we studied mitochondrial parameters in cell lines, and we even tried the effect of some drugs. If you want to check, and if you want more advice or help you can contact the lab of Prof. Julio Montoya in Zaragoza, Spain where I did my PhD. They are experts in this field.

http://www.ncbi.nlm.nih.gov/pubmed/23223015

http://www.ncbi.nlm.nih.gov/pubmed/22561905

http://www.ncbi.nlm.nih.gov/pubmed/21828074

http://www.ncbi.nlm.nih.gov/pubmed/20566709

Dear Eugenio,

I agree with Michael Duchen that keeping things simple is the best approach, at least in the beginning. However, if you are really want to measure mtDNA damage (and I am not sure that this should be, necessarily, a first step), there are no really simple techniques that give you biologically relevant results. If you have enough biological material, I would suggest you tried to use analytic techniques, such as micro-HPLC couple with electrochemical detection. This technique allows for (relatively) simple determination of 8-OH-dG, but requires a large amount of isolated DNA. If your experimental model does not allow you to isolate such large quantities of DNA, the long-extension PCR technique, developed by Janine Santos and Ben van Houtten, is very good, with the caveat that it only detects polymerase-blocking lesions. There is a lot of literature on both techniques, and if you are interested I would be happy to send you some.

Best,

Nadja Souza-Pinto

Re: Ruslan. I agree, there is a lot of alternative approaches. I just happened to have experience with the Hanzatech Oxytherm system (reasonable price, durable, but sensitivity and electrode as well as sample handling issues), the Seahorse (fantastic system with high throughput plate format, but limited parameters to measure; i.e. OxPhos and deltapH for glycolysis), the WPI Apollo free radical Analyzer (excellent for chemistry experiments but terrible with biological samples). The Oroboros, with the new pH and fluorescence modules as well as the TPP and NO electrodes allows you to simultaneously measure oxygen consumption, transmembrane potential, H2O2 release, NO, Ca, etc.

Dyes-based ROS measurements are very problematic and yield very qualitative results at best! I routinely use electron paramagnetic resonance spectroscopy to monitor ROS release from mitochondria and live cells. This virtually the only technique capable of detecting, identifying, and quantifying ROS.

Awesome! thanks to everyone for all the advice. A lot of information to process!! You're all great :-)

Your question is quite interesting...

And the answer will very much depend on the link between laboratories and compagnies... Since each one has choosen its own system to screen cells.

Indeed you may use different system... from the most

• Classical oxygen electrode Like the Hansatech oxyterm system for the measurement of the electron transport chain (Clark electrode) which could be adapted to link a measurement of the proton efflux from mitochondria with a pH electode and also to accept a system based on a tetraphenylphosphonium (TPP+ or derived system like TMPD+) electrode to simutaneously measure the mitochondrial membrane potential. A system which is very perfomant but heavy to handled since you may be expert to set up all the conditions together (but this is possible). After that it will be nice to run this type of system routinely

• The new Oroboros suystem, with the new pH and fluorescence modules as well as the TPP+ and NO electrodes allows you to simultaneously measure oxygen consumption, transmembrane potential, superoxide anions, H2O2 release, NO, Ca, gluthation, cathepsin, NAD(P)H fluorescence etc. effectively. This is a quite expensive investment and needed also a strong expertise.

• You can of course experiemnted the Seahorse system... Which is certainely the best for screening but expensive.

• And effectively, The BIONASS technology which combine the analysis of cells on a support where you can measure the detachment of the cells (impedance changes) together with the oxygen consumption and an enzymatic activity. It is only a bit difficult to get a demonstration in your laboratory since this is a small compagny.

• Don't forget about flow cytometry which will allow you to test the cellular metabolism on live cells, i.e. calcium pulses, mitochondrial membrane potential, ROS production (measurement of superoxide anion and H2O2 production), and eventually the fact that cells may be push to undergo apoptosis or necrosis by probes which affect the mitochondrial bionergetic (from that you can get a I50 index).

• As somes others have tell you, do not forget about basic biochemistry.

The study of the mtDNA will be certainely useful to.

Patrice

Lots of good answers there thus far. If you are interested in measuring mt protein levels in treated versus untreated cells, I would suggest a SILAC approach. Please see the following publication for more information:

http://www.nature.com/nprot/journal/v1/n6/full/nprot.2006.427.html

Oroboros instruments are very useful in mitochondria study. In my opinion it is one of the best apparatus ..... . It is expensive but there are a lot of laboratories worldwide which have it/them - these scientists who have these oxygraphs are able to cooperate - so, if somebody does not possess oxygraph can develope a cooperation with others. It is really easy :)

I work with using Oroboros oxygraph about 3 years - so I feel experienced in some technics described at www.oroboros.at and I have the will to start common works :)

so feel free to contact with me :)

my e-mail address: [email protected]

All of the above mentioned comments are pretty good another thing that you can measure is alteration of glucose uptake and lactate production which can be a complimentary assay for oxygen consumption. I am not sure what kind of cell lines you are using but if you want to evaluate the effect on cancer cells these can be a good piece of information to have.

Dear Eugenio,

We are working at Santiago de Compostela with high-resolution respirometry (Oroboros). Routinely we perform assays to study the respiration and the interaction of compounds with ETS complexes. Also, we have the possibility to measure membrane potential with a TPP electrode. If you are interested do not hesitate to contact us.

For ROS generation you can use Amplex Red or DCF2DA method

for amplex red ref: http://products.invitrogen.com/ivgn/product/A12221?ICID=search-a12221

for DCF2DA method ref: http://www.sigmaaldrich.com/catalog/product/sigma/35845?lang=en®ion=IN

Alamar blue assay (resazurin salt) can be used as a measure for the mitochondrial respiration and is also used as an empirical assay for cell viability.

Ref: http://www.sigmaaldrich.com/catalog/product/sigma/r7017?lang=en®ion=IN

Interesting informations above, and want to add here such as Mitochondrial dynamics can be studied using organell lights or simply using mitotrackers. In case of mitochondrial ROS mitoSOX Red would be the best choice.

Mahendra, could you send or indicate the paper in which I will be able to read the protocol for mitoSOX Red mesurements? I am really interested in using this method for ROS studies in mitochondria.

thank you in advance, Magda

http://www.ncbi.nlm.nih.gov/pubmed/21747057

Good luck!

If you have further question, you are welcome!

Thank you so much :)))) !!!!

http://www.seahorsebio.com/

These people have also got pretty good machines to study what you asked for Eugenio!

And their online tutorials are quite good. You need to create a profile to access some of them but it is easily done!

Good luck!

If you want to study mitochondrial metabolism in cell culture, it is important to first consider the metabolic state of the cells. Most cells grown in glucose-containing medium are predominantly glycolytic (the Crabtree effect), and are therefore not a good model for studying mitochondrial metabolism.

Here is a paper which describes a method for switching the metabolic state of cultured cells from glycolysis to OXPHOS.

Article Circumventing the Crabtree Effect: A method to induce lactat...

I'm looking for an assay/ method to test the changes in trans-membrane transport in mitochondria. Any suggestions will be helpful. Thanks!