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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