You wrote We are attempting to assess apoptosis in a transfected cell line in which we can not use annexin (the cells are far too adherent, and by the time they are harvested, annexin binds with an equally high affinity to all cells). We would like to use mitochondrial membrane potential, but need an indicator that does not obscure our GFP signal (i.e. excited by the 633 nm diode on our Calibur). Previously I had seen people use DiOC6(5), but Molecular Probes no longer offers this product. Molecular Probes suggested we try DiS3(5), and I wondered if anyone had used this indicator with any success, or if there were any other suggestions for 633 excitable, >670 emitting indicators?? Thanks in advance for any replies, Keith Bahjat Graduate Assistant University of Florida College of Medicine kbahjat@ufl.edu We are supposed to be specialist of mitochondrial membrane potential adn as far I understand your problem, you can certainely use 488 nm excitable dyes like CMXros (chloromethyl X rosamine, Mole. Probes) which fluorescence in red (providing you assume a proper calibration with you cell type and with a decoupling agent for being sure that this really the mitochondrial membrane potential you measure). You may use this dye in the the range of 1 nM to 50 nM max. in order not to be cytotoxic for the ATPase and the respiratory chain. The other possibility is simply to use TMRM (tetramethyl rhodamine methyl ester) which more frequently used in confocal but works well in flow. I am also working with such problems and we might be able to collaborate if you are open to my proposition. I am more expert in mitochondrial bioenergetic than in trasnfection and can provide you some help in this field. But if I look at the discussion from Shapiro... and I totally desaggree with the use of Rh123 at 10 uM or any of such way of thinking... Just forget about that... "for mitochondrialmeasurements, you'd probably want to use the dye the way rhodamine 123 was originally used, i.e., by loading cells with dye at an intial concentration of 10 uM or more, and then washing before making measurements. Cells with energized mitochondria should retain the dye, while those with deenergized mitochondria (apoptotic cells) should not. You'll probably have to play around with dye concentrations and timing" Sincerely yours Dr. Petit Patrice X. 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 - 11:53:15 EST