Douglas P. Olson wrote: We stain mitochondria (we've used mitotracker red) and then isolate them by a simple cell lysis procedure which keeps them viable but removes them from the cytoplasm. Then, we sort to isolate pure mitochondria (to get rid of all the other organelles and cell wall remnants). However, the mitochondria don't seem to stay alive. We've considered that they may just get too beat up when sorted, but has anyone else tried a similar expt. (sorting organelles) and either failed or (even better) gotten it to work? ----------------------------------------------- Douglas P. Olson Experimental Hematology/AIDS Research Center Massachusetts General Hospital Harvard Medical School 149 13th Street Boston, MA 02129 (617)724-2668 - Phone (617)726-4691 - Fax Dolson4@partners.org - Email Dear Doug, I am sure that you would find informations in the two publication below. I have also seen the answer of Howard... OK, he is back into the problem of cationoic dyes and respiartory inhibition which is by far too much under discussion. Rottenberg has answer part of the question in his publication and to what concern our work we alway use a less duye as possible ... in the range of 0.5 to 2 nM. Most of the publication on cell death and mitochondria cited our early papers and reproduced mistake in the writting, at least in my own publication. But concerning your work, I believe that the problem come from the fact that the resuspension medium is inadequate. I am actually publishing a paper on isolated mitochondria and flow cytometry which clearly analyse the relation between mitochondrial respiration and mitochondrial membrane potential of functional mitochondria in the clark electrode and in flow. I can assure you that the mitochondria stay functional after sorting.... 1. Petit, P.X., P. Diolez, P. Müller, and S.C. Brown. 1986. Binding of concanavalin A to the outer membrane of potato tuber mitochondria detected by flow cytometry. FEBS Lett 196: 65-70. 2. Petit, P.X., J.E. O'Connor, D. Grunwald, and S.C. Brown. 1990. Analysis of the membrane potential of rat- and mouse-liver mitochondria by flow cytometry and possible applications. Eur. J. Biochem. 220: 389-397. 3. Rottenberg, H., and S. Wu. 1998. Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells. Biochim. Biophys. Acta 1404: 393-404. You may find information in the two publication below. 1. Petit, P.X., M. Goubern, P. Diolez, S.A. Susin, N. Zamzami, and K. G. 1998. Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: the impact of irreversible permeability transition. FEBS Letters 426: 111-116. 2. Marzo, I., S.A. Susin, P.X. Petit, L. Ravagnan, C. Brenner, N. Zamzami, and K. G. 1998. Caspases disrupt mitochondrial membrane barrier function. FEBS Letters 427: 198-202. Anyhow, if you e-mail me we can certainely solve the problem. Patrice \ | / (o o) \ | / _______________oOo_____oOo_____oOo_ (o o)__oOo________________ Dr. Petit Patrice X. Institut Cochin de Génétique Moléculaire Team "Apoptosis and mitochondria" INSERM U129 - CHU Cochin Port-Royal 24, rue du Faubourg Saint-Jacques F-75014 Paris, France. Tel: 33 01 44 41 24 11 Fax: 33 01 44 41 24 21 E-mail: pxpetit@icgm.cochin.inserm.fr € This e-mail is confidential and should not be used by anyone who is not the original intended recipient. If you have received this e-mail in error please inform the sender and delete it from your mailbox or any other storage mechanism €
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