Ty Lee mentioned FITC-VAD-FMK usage for detection of apoptosis. I am
wondering if it is known whether this binds to activated caspases. What is
the commercial source for this? Finally, can it be used in a flow cytometry
assay?
A note of caution about solely relying on activated caspase-3 and/or
PARP as indicators of apoptosis. Recent studies have shown that caspase-3
and PARP are activated in nonapoptotic proliferating human peripheral blood
T cells in vitro. We have confirmed that caspase-3 is constitutively
activated in human T cells that have been activated with allogeneic
antigens. However, Fas-mediated apoptosis does not accompany enhancement in
caspase-3 activity. All these observations were made in human T cells. This
may or not be the case in neuronal or other type of cells. The following
references may be helpful:
1. Miosse et al. 1997. J. Biol. Chem. 272: 13459.
2. Wilheim et al. 1998. Eur. J. Immunol. 28: 891.
3. Alam et al. 1999. J. Exp. Med. 190: 1879.
Jay
Sundararajan Jayaraman, Ph.D.
Research Assistant Professor
Dept. of Pathology
University of Miami Medical School
DRI Building Room 5018 (R-134)
1450 NW 10th Avenue
Miami, FL 33136
Phone: (305) 243-6100
Fax: (305) 243-4553
> -----Original Message-----
> From: tylee [SMTP:tylee@itis.com]
> Sent: Thursday, March 02, 2000 11:01 PM
> To: Cytometry Mailing List
> Subject: FITC-VAD-FMK & Apoptosis Markers / Re: apoptosis in neuronal
> cells
>
> 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
> <mailto:MONIQUE.VANHOEK@roche.com>>
> To: Cytometry Mailing List < cytometry@flowcyt.cyto.purdue.edu
> <mailto: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
> <http://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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