ANSWER TO X-Sender: butterfield.15@pop.service.ohio-state.edu If anyone has used the mitochondrial membrane probes such as JC-1 or MitoTracker can you tell me what works as a positive control? Thanks. Dear Butterfield, At first, take care to use low probe conecntration, and this is the case for all the probes. inthe nM range will be the best. The control is often to collapse to mitochondria membrane potential with decoupling agents which could be FCCP or mClCCP (sigma). You may realize a titration from 1 micromolar up to sometime 50 micromolar depending of the cell type and of the medium used because the FSC in the medium or the others proteins will trap part of the decoupling compounds. See first Rottenberg et al. (1998) and our proper publication taking into account that the amount of probe has to be reduced as much as possible up to 1nM. 1.Rottenberg, H. & Wu, S. Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells. Biochim. Biophys. Acta 1404, 393-404. (1998). 2.Petit, P.X., O'Connor, J.E., Grunwald, D. & Brown, S.C. Analysis of the membrane potential of rat- and mouse-liver mitochondria by flow cytometry and possible applications. Eur. J. Biochem. 220, 389-397 (1990). 3.Zamzami, N. et al. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181, 1661-1672 (1995). 4.Petit, P.X. et al. Alterations of mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J. Cell. Biol. 130, 157-167 (1995). Sincerely yours Patrice \ | / (o o) \ | / _______________oOo_____oOo_____oOo_ (o o)__oOo________________ Dr. Petit Patrice X. Institut Cochin de Génétique Moléculaire 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
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