Dear flower... Effectively, HeLa cells have few mitochondria. The micromolar concentration of the dye you use is to high anyway... but it is not the reason. Is you lower the concentration of the probe to nanomolar concentration you should avoid any cytotoxic side effects do to the inhibition of either the respiratory chain and/or of the FOF1 ATPase. Anyhow, filter your dye solution to discarb the dye aggregates (Red) and determine the solution concentration after filtration. The best control of your experiment will be realized with an decoupling agent like FCCP or CCCP. You may have to titrate the decoupling agent in fonction of the composition of the culture medium (espacially in fonction of the amount of FCS). Rage of values 1 to 50 micromolar. We work routinely with HeLa cells and mitochondrial membrane potential without problems. You can also use the dye DiOC6(3) at 1 nM (15-30 min incubation) in order to make free the red channel to determinedthe viability of your cells wit PI. Two references: 1. Susin, S.A., N. Zamzami, N. Larochette, B. Dallaporta, I. Marzo, C. Brenner, T. Hirsch, P.X. Petit, M. Geuskens , and G. Kroemer. 1997. A cytofluorometric assay of nuclear apoptosis in a cell-free system: application to ceramide-induced apoptosis. Exp. Cell Res. 236: 397-403. 2. Petit, P.X., H. Lecoeur, E. Zorn, C. Dauguet, B. Mignotte, and M.L. Gougeon. 1995. Alterations of mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J. Cell. Biol. 130: 157-167. 3. 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. I am ready to answer more questions if necessary. 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
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:54:06 EST