Hello All, Try blocking your cells with Rabbit serum. This was the advice from Mark G. Edinger, Tech Application Specialist, Pharmingen. It's supposted to improve the separation. But if your not going to use rabbit serum, the seperation of positive to negative will be low, so place your negatives just below 10e2. I've only run Caspase 3 once, and it wasn't blocked. Lora Barsky Children's Hospital Los Angeles > -----Original Message----- > From: Candace Enockson [SMTP:enockson@musc.edu] > Sent: Wednesday, September 08, 1999 12:43 PM > To: Cytometry Mailing List > Subject: Re: caspase-3 > > > I have a client who has used this in virally infected cells with some > success. However,there is not very good separation between negative and > positive. I was told by a Pharmingen person that this was a problem with > this particular lot of antibody. Also, it does have a lot of non-specific > background over non-stained cells. Hopefully they will be coming out with > the monoclonal version soon. > > Candace Enockson > Medical University of South Carolina > > > --On Tue, Sep 7, 1999 9:35 AM -0400 Maryalice Stetler-Stevenson > <stetler@box-s.nih.gov> wrote: > > > > > We also have tried this antibody in parallel with Annexin V without > > success but haven't done a time course, etc to determine if there are > > biological reasons this isn't working for us. I would be interested in > > others experience with this antibody and any tricks people may have used > > to get it to work better. > > > > Maryalice > > > >> Dear all > >> > >> We have been using a anti-Caspase-3 rabbit polyclonal ab conjugated > >> with PE (this is supposed to bind to the active form only) by flow. > >> We've looked for caspase-3 activation in camptothecin or etoposide > >> treated Jurkatt cells (cytospins confirm apoptosis in these > >> cells) after fixation and perm, without success. This ab is from > >> Pharmingen. > >> > >> Does anyone have experience with looking for caspase-3 activation by > >> flow ? Are there other antibodies available ? > >> > >> Any comments would be much appreciated. > >> > >> Fran Gibson > >> Dept Haematology > >> St. George's Hospital Medical School > >> London > > > > Maryalice Stetler-Stevenson > > Director Flow Cytometry Unit > > Laboratory of Pathology, NCI, NIH > > >
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