I appreciate your comments, Derek. My colleague is working with adherent cell lines and is attempting to stain with Annexin V kit from BD. However, as you say the trypsin is causing some problems. Does anyone have any experience using adherent cells lines with Annexin V to analyze apoptosis? Can you tell me what you use in lieu of trypsin and any other tricks to the protocol to make it work well? I appreciate your assistance. Rick -----Original Message----- From: Derek Schulze [mailto:flow@post.queensu.ca] Sent: Thursday, March 15, 2001 12:21 PM To: cyto-inbox Subject: RE: Apoptosis -- no direct responses Two things here: You are more likely to get responses to your question if you ask more specific questions. If you want to work with Annexin V first go to the literature and see what has been done with your cell type. Some key things to think of to begin with though: -AV works best with non adherent cell lines because trypsinizing can expose PS on the outer plasma membrane leaflet giving false positives. -think of a good positive control that is appropriate for your cells (ceramide treatment, serum starvation or anti-FAS etc...) -optimize your concentration and exposure time for whatever method if inducement you are going to use so as to minimize necrosis -if it is appropriate confirm your outcomes with a fluorescence microscope (only really good for later apoptosis - nuclear fragmentation) -AV is generally used for very early apoptosis signals, for more precises results you might consider doing a tunnel assay (tdt labelling of DNA fragments with BrdU-FITC) for slightly later apoptosis events. ----------------------------------------------- People not responding directly to questions may have to do with the fact that when the reply button is hit the email from the postee is presented NOT the mailing list. This should be changed so that the mailing list is presented first. I have to hit "reply to all" in order to get the mailing list address to show up. Maybe this has something to do with how I have Eudora set up but I can't see anything obvious. > > On Mon, 12 Mar 2001, Ashman, Margarita wrote: > > > > > > > > Does anyone have experience with annexin V for running apoptosis > > > > > > Thanks > > > > > > Margarita Ashman > > > mashman@med.miami.edu > > > > > Derek Schulze Cancer Research Labs Queen's University 353 Botterell Hall Kingston, ON, K7L 3N6 (613) 533-6635 http://meds.queensu.ca/medicine/crl/
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