We have stained adherent cells with Annexin V. Gibco/BRL sells a non-enzymatice media for detaching adherent cells. We found that worked well. Maryalice >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/ Maryalice Stetler-Stevenson Director Flow Cytometry Unit Laboratory of Pathology, NCI, NIH "Learn the rules so you know how to break them properly." The Dalai Lama
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