Annexin and intracellular staining

Marcelo.Decarvalho@chu-fortdefrance.fr Marcelo.Decarvalho at chu-fortdefrance.fr
Thu Nov 24 09:44:23 EST 2005


Dear all,

We are studying the pro-apoptotic action of a drug on viral infected CD4
cells in vitro. Not all cells are infected, and we would like to assess
the drug-induced apoptosis on infected vs non-infected cells. I was
therefore wondering how to do annexin and viral intracellular protein
co-staining on CD4 T cells and I would like to submit some questions to
the experts :

Which step to do first after the initial cell surface labelling, annexin
or intracellular? Indeed, I suppose the fixation/permeabilization step
would cause PS expression on outer cell membrane, rendering all cells
Annexin positive. If it's true, the annexin step should precede the
intracellular one. However, the annexin staining is dependent on calcium
and reversible. It would therefore be lost in the following fix/pem
steps. Is it right? If so, a possible solution would be doing all
fix/perm steps and washes on 1x annexin buffer (in the presence of
calcium).

Otherwise, doing the fix/perm steps before would result on annexin
staining of internal PS. Again, if it's true, a possible solution would
wash out perm buffer and let cells in buffer without saponin (how long?)
to "close" all membrane pores...

I would appreciate your comments on this points and other potential
solutions to this issue.

Many thanks for your help,

Marcelo
 
-------------- next part --------------
A non-text attachment was scrubbed...
Name: BDY.RTF
Type: application/rtf
Size: 1510 bytes
Desc: not available
Url : https://lists.purdue.edu/pipermail/cytometry/attachments/20051124/052564cf/BDY-0002.rtf


More information about the Cytometry mailing list