Rita, my experience with Annexin in various cell situations is that you will get a fair number of baseline positive cells for annexin and PI which are cells that have already "died" either from necrosis or apoptosis. If they are only annexin positive the implication is that they are apoptosing on their own, without any induction. Do you get more annexin after you induce or is Deamthasone the only thing you've tried so far? In either one of the above scenarios, this baseline positivity must be taken into consideration with appropriate quadrant placement so that accurate measurement of positivity greater than this baseline can be shown. In other words, this is your cutoff control. Sorry, I can't help you on the second part of your question - I've not done human PBMCs. Candace Enockson Flow Cytometry Shared Facility Medical University of South Carolina --On Fri, Apr 23, 1999 9:45 AM +0000 Rita Cavaleiro <ritacavaleiro@hotmail.com> wrote: > > I am starting to use Annexin-V method to measure apoptosis. After > labelling the cells with annexin and PI, I add the binding buffer to > the cells without any wash and acquire immediately, in agreement with > the annexin-FITC staining protocol from Pharmingem. With this protocol > we obtain, unexpectedly, a lot of positive cells for annexin in > unstimulated human PBMCs in culture for 24 hours. I am not sure if > this high level represents unspecific labelling because there is no > wash after the adition of annexin. None of the protocols use this > wash. Could anyone tell me if there is any problem in doing this wash? > In addition, I am not sure about what control I have to use to put > the cut off. What is the best positive control to induce apotposis in > human PBMCs and in what concentrations ?. I tried Dexamethasone but it > did not work. Can anyone help me? Thanks in advance. > > > > Rita Cavaleiro > Cellular Immunology Unit > Faculty of Medicine of Lisbon, Portugal > e-mail: ritacavaleiro@hotmail.com > Fax: 351 1 7951780 > > > ______________________________________________________ > Get Your Private, Free Email at http://www.hotmail.com
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