Snezna,
Another approach you might try is to use FITC-VAD-FMK which is a fluorescently labeled cell permeable peptide inhibitor of caspases. FITC is the fluorescent tag; VAD is valine-alanine-aspartic acid, and FMK is fluoromethly ketone (the irreversible protease inhibitor "business end" of the molecule. The FITC-VAD-FMK can enter live cells undergoing apoptosis and irreversibly bind to active caspase enzymes.
I am not sure what GD 12-14 cells are or how they will be removed from fetal brain samples; however, for another approach, I might recommend trying immunohistochemistry as an alternative to flow. There are a few marker antibodies that are becomming available to stain active caspases or the protein fragments that result from their protease activity. I have done immunocytochemistry on human cells using an antibody from Promega that is specific for the cleaved form of PARP (i.e. a marker of apoptosis); but I'm not sure if it will work on rat neuronal cells. They also have an antibody against the active form of caspase-3 that should be much more species cross reactive because of the conserved amino acid sequence of that protein. Caspase-3 activity has been shown to be an earlier marker than Annexin-V staining because caspase activity is required for the phosphatidyl serine translocation to the outer membrane that Annexin V binds to.
My two cents worth.
Ty Lee
-----Original Message-----
From: Vanhoek, Monique {BERK~Berkeley} <MONIQUE.VANHOEK@roche.com>
To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
Date: Thursday, March 02, 2000 5:47 PM
Subject: RE: apoptosis in neuronal cells
Yes, we (Roche Molecular Biochemicals) offer many kits for studying apoptosis. The Annexin-V kits are designed to be done by FACS. I reccommend the Annexin-V staining kit and the Alexa dye. See http://biochem.roche.com/apoptosis/prod07.htm for more details. You can order by phone (1-800-262-1640) or at www.IbuyRMB.com
-----Original Message-----
From: Snezna Rogelj [mailto:snezna@nmt.edu]
Sent: Tuesday, February 29, 2000 4:04 PM
To: Cytometry Mailing List
Subject: apoptosis in neuronal cells
Wise (wo)men of Flowland -
A colleague of mine would like to know if flow cytometry can be used to determine whether a subset of fetal brain cells (rat, GD 12-14) is undergoing apoptosis. Yes? No? Any and all information is bound to be useful.
With many thanks,
Snezna
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