This is following up on Howard's suggestion about DiIC1(5) as a mitochondrial membrane potential probe to detect apoptosis. In our hands this gives a large decrease in fluorescence following loss of mitochondrial membrane potential, either during apoptosis or in response to uncoupling agents. Because it is red HeNe excited, it should be spectrally compatible with GFP. Our staining protocol for DiIC1(5) is 40nM for 30 min at 37°, excite red HeNe, collect 675nm. Separation of low MMP cells is greater if you resuspend in dye-free medium. However, we rarely bother to do this, as we usually see daylight between high and low MMP populations without resuspending. We have published several papers using this stain. Most recent (and technically detailed) is Sheng-Tanner et al., Radiation Research 1998;150:636-647. Molecular Probes were thinking of discontinuing DiIC1(5) last time we ordered some (late 1998), so you might be out of luck. David Hedley Ontario Cancer Institute Toronto > -----Original Message----- > From: Howard Shapiro [SMTP:hms@shapirolab.com] > Sent: Monday, March 15, 1999 8:32 PM > To: Cytometry Mailing List > Cc: Cytometry Mailing List > Subject: Re: Mitochondrial Membrane Potential > > > Keith Bahjat writes: > > >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?? > > > > In most circumstances (including the cuvette measurements for which > DiSC3(5) > has been so widely used), DiIC1(5) (hexamethylindodicarbocyanine iodide), > which I believe Molecular Probes also sells, is a better dye. DiSC3(5) is > more toxic to cells and decomposes faster, especially in dilute solution > (maybe that's why Molecular Probes recommended it...); it also excites > less > well than DiIC1(5) at 633-635 nm. I frankly don't see why anybody uses > DiSC3(5) anymore. > > The chromophore in DiIC1(5) is the same as that in Cy5. Because DiIC1(5) > has methyl side chains instead of the propyl side chains in DiSC3(5), it > is > slightly less hydrophobic, but that shouldn't make a big difference. I > have > used DiIC1(5) for measuring cytoplasmic membrane potential in mammalian > cells and for measuring bacterial membrane potential; for mitochondrial > measurements, 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. > > Also note that DiIC1(5), like all other lipophilic cationic "membrane > potential indicator" dyes, will be pumped out of cells by the glycoprotein > drug efflux pump. > > -Howard
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