That will be tricky. We previously tried to do FACS using freshly isolated mitochondria and it worked. Frozen samples won't work in this method.

we are also using fresh isolated mitochondria . what kind of antibodies you used? can you tell me? can you give me any references?

Thanks

Hi Khare,

it is possible to perform this kind of experiment. I know a boy that performs this kind of experiment but with mitochondria coming from mice that stably express mito-GFP (unfortunately I hasn't yet publish his study... and the method ). However to study the mitochondrial changes in structure and size after viral infection (or other stress event) you could use a different approach . why do you want use facs for this kind of analysis? Moreover, why do you want to perform the analysis on isolated mitochondria and not directly on your cells with a mitochondrial specific dye ? and a last question.. why do you speak of "lysate mitochondria"? Do you isolate mitochondria and then you add a detergent?

regards

Francesco

Hi Francesco,

Thanks for the reply. We are also using mitotracker to study the same thing in cells as well as WB too. But would also like to try this method. I am doing in cell lysate and also in enriched mitochondrias. we are trying to find an antibody which will help us identify the change in structure /morphology of mitochondria before and after viral infection. Can you suggest anything ? any antibody or any other method........

thanks again

meenakshi

Hi meenakshi

For wb you can use an antibody against opa1 or drp1. These two proteins are essentials in mito-dynamics. Do you know them? If the viral infection is a stress event probably you will induce mitochondrial fragmentation and so you should observe a change in opa1 processing ( that you can easily see with an sds page 7%) or the ri-localization of drp1. To verify this last case you must prepare the cytosol fraction and the mito-fraction, if your treatment induces fragmentation trough drp1 pathway, you will see the protein moving from the cytosol to mitochondria. However in literature you can find a lot of papers that use these two proteins to study the mito-morphology.

Moreover if you want to study changes in mito-structure ( as cristae organization) you could evaluate the activity of the complexes or their quality with B/N PAGE.This could be an indirect way but can give you a lot of information on the healthy of your mitochondria pre and post infection. In the end, the best way to perform your study on the changes in mitochondrial structure is the electron microscopy. I'm not a big expert in FACS but I read enough literature about mito-morphology and I never read Facs as methods.. But you could be the first ;).

I hope that these answers could be useful for you. If you need other information write to me without any problem.

Regards

Francesco

Hi Francesco

Thanks again. We are using WBfor drp1 both in cell lysate and mito fraction, and you are right there are no papers on FACs for mitochondria and I could be the first one :). Unfortunately its me who have to do that work. I am way past my post doc time and now working in senior level as assay development person so it falls on me .......:) The post docs in lab are working on WB and imaging assays,,,,,,,:) me the lucky one got this one :) thats why I am sending these questions to any body who can help........

Will try BN PAGE though........we are so far not going to do EM but may be in future.

thanks for your help

meenakshi

No problem Meenamshi

However for you scope you need to have a machine that is able to read very small events.. As dye you could use TMRM. if you lysate the cells and isolate mitochondria you'll destroy the network organizzation that is more important compare to the shape. So i don't understand in which way facs in isolate mito could help you but i'm only a phD student .. :) I think that is unusufull to say but...don't fix the cells with para if you want to use the cells.. Perform your assay with live cells.... However good luck for your study..

Regards francesco

Good morning everybody,

Nobody talked about Electron Microscopy !!! This is probably the BEST technic to see size and morpholoy...

Regards

Nath

Dear Nath,

thank you for your answer but probably you miss the previous answers. We have already speak about EM. However I don't agree that EM is the BEST technic to see mitochondrial size and morphology for at least three reasons.

First: EM is an expensive technique and so it is very difficult to obtein quantitative results.

second : with EM you could have changes in morphology linked with the sample preparation.

third: Mitochondria, as you perfectly known, are a network with a 3D organization and with EM you could only have a 2D picture, losing part of the information as consequence of the cut. So for me the best technique for this kind of experiment is live imaging, with a Z-stack acquisition followed by 3D reconstruction.

On the other hand EM is the only technique that we have to see the crestea organization.

Regards.

Francesco

Dear Meenakshi,

today I found this paper and I thought at this your old question.

regards,

Francesco 

http://www.ncbi.nlm.nih.gov/pubmed/23080405