gfp/apoptosis caveat

Chris Groves (chris_groves@msmail.mpi.com)
31 Jul 1997 08:46:13 -0400

Annexin v caveat...annexin v has been shown to have calcium dependent
binding by Manon van Engeland et al (Cytometry 24:131-139, 1996) and =
others. Many commercial vendors sell annexin v, but in particular =
Pharmingen sells annexin v pe. Just make sure calcium ions are present =
for binding to cells and that ca++ remains in media when analyzing.
Chris

A few small additions to Mario's note. First, annexin V biotin is also
commercially available and indirect staining may be easier than trying to =
do a
conjugate. Second, in my experience annexin V staining will not survive =
PFA
fixation unless some divalent cation (calcium) is present in the fixative =
(and
any wash steps!). I have only used bovine annexin V, but I assume that =
this
will be true of human recombinant annexin V also.

Tom Frey

We haven't specifically tried GFP with annexin, but there is no reason =
for this
not to work--we often use GFP with a wide variety of fluorochromes, like =
PE,
Cy5PE, TR, APC, etc; annexin V can be conjugated to any of these to be =
used in
combination with GFP. If you have the annexin V protein (it can be =
obtained
both commercially and in collaboration with some scientists producing
recombinant versions), conjugation instructions can be found at
"http://cmgm.stanford.edu/~roederer/abcon/".

BTW, the annexin V staining survives 0.5% paraformaldehyde fixation just =
fine.

mr

> Has anyone tried this combination? Or are there any other
> apoptosis markers that would work with GFP?