Apoptosis detection with Annexin V and DiOC6 -Reply

Tue, 21 Jan 1997 08:03:07 -0500

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

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