We have done quite a lot of work on this recently. Mostly we have used DiIC6(5) to measure mitochondrial membrane potential, since this excites with a red HeNe laser and allows simultaneous measurements of annexin V-FITC and indo-1. We use 40nM DiC6(5) for 30 min at 37*, and add annexin V for the last five min. We add 5?g/ml PI as a live/dead discriminator at the same time as the annexin V. The DiIC6(5) protocol is essentially the same as for the more widely used DiOC6(3). Results for ara-C induced apoptosis in leukemic blasts shows that loss of mitochondrial membrane potential and phosphatidylserine exposure occur simultaneously, and precede an increase in ionized calcium. This is slightly different from the results of Castedo et al (J immunol 1996;157:512-521), where annexin V positivity occurred about an hour after the mitochondrial membrane changes. You should check the Castedo paper, since they did dual labelling of annexin V and mitochondrial membrane potential off an argon laser, using the new dye CMXRos (available from Molecular Probes). Martin Poot (who will no doubt also respond to this message) has a recent paper in J Histochem Cytochem that give more detail about CMXRos. Hope this helps David Hedley Ontario Cancer Institute/Princess Margaret Hospital Toronto
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