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Yevgeniy,

Uncouplers like CCCP, FCCP, DNP can block mitochondrial ATP synthesis 
by collapsing the proton gradient, but as Tobias and Carl point out, 
many intracellular processes will quickly come to a halt as ATP 
concentrations are consumed. Choosing a cell type that has a high 
membrane impermeability, reducing the necessity of ATP pumps, and 
supports a functional glycolytic pathway would seem to be crucial. 
You might be able to drop the temperature to slow down metabolism as 
well. To obtain meaningful data it would seem that you should monitor 
a number of physiological parameters simultaneously including cell 
volume, intracellular Ca++, PM potential, etc

Mario


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>Greetings,
>	While the tenor of Carl's remarks is right, keep in mind that 
>tissues differ in their ability to live aneorobically. Some 
>tissue/organs can survive hours without oxidative phosphorylation. 
>Of course this will induce various changes in the cell physiology 
>but these can stop short of death. I don't know about KCN but there 
>are certain uncouplers (e.g., dinitrophenol) that will stop oxphos 
>without directly affecting other componenets. I think oligomycin is 
>another. I expect that membrane potential would decrease within 
>minutes so add mitotracker at that point and see. Not the greatest 
>experiment in the world but could serve your purpose.
>
>	As ever
>		Tobias Baskin
>
>>Because tissue function depends on cell function that makes up the 
>>tissue, and because mitochondria are intracellular, treating tissue 
>>without treating cells is not possible.  Moreover, poisoning 
>>mitochondria with cyanide, which will certainly stop their 
>>function, will quickly kill the cells/tissue, so tissue function 
>>would stop. Because the mitochondrion is so fundamental to the 
>>workings of a cell, I don't see a way out.  I'm trying to imagine a 
>>fully functional  eukaryotic cell without mitochondria.  You could 
>>certainly treat with cyanide, then see how Mitotracker stains, but 
>>the tissue will likely not be functional at the same time.
>>
>>C
>>
>>Carl A. Boswell, Ph.D.
>>Molecular and Cellular Biology
>>Univ. of Arizona
>>520-954-7053
>>FAX 520-621-3709
>>
>>
>>-----Original Message-----
>>From: Confocal Microscopy List 
>>[mailto:[log in to unmask]] On Behalf Of Yevgeniy 
>>Romin
>>Sent: Tuesday, January 18, 2011 8:51 AM
>>To: [log in to unmask]
>>Subject: Stopping Mitochondrial Activity in whole mount organs
>>
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>>
>>Hello everybody.  This isn't exactly a microscopy question, but 
>>maybe someone here has experience with this issue.
>>
>>We are doing experiments involving assessing the viability of cells 
>>in whole mount live organs (liver, in this case), and we are using 
>>Mitotracker in order to see whether the cells are alive.  Lately we 
>>have been doubting the integrity of our Mitotracker staining, and 
>>we would like a negative control to work with.  Does anybody have 
>>any experience with stopping the mitochondrial activity in live 
>>tissues, like anything that we could inject into the organ?  We 
>>need these negative controls to be on tissue, not on cells.  Any 
>>advice you can give will be greatly appreciated.
>>
>>---------------------------------------------------
>>Yevgeniy Romin
>>
>>Digital Microscopist
>>Memorial Sloan-Kettering Cancer Center
>>Molecular Cytology Core Facility
>>1275 York Ave. Box 333
>>New York, NY 10065
>>Tel.646-888-2186
>>Fax. 646-422-0640
>>---------------------------------------------------
>>
>>
>>
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-- 
________________________________________________________________________________
Mario M. Moronne, Ph.D.

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