Hi Jose, your query is very similar to issues that I raised through the group, in Feb 2000. Hopefully, you might find some of the issues discussed there helpful. If you look at the archives for around 10-21st Feb, 2000, there is a lot of discussion. I posted a summary of people's responses on 21st Feb. I am sure that you will get many answers to your queries from people much more expert than me, but I will maybe just 'chip in' with an opinion on the questions you posed: 1) Annexin (AV) is apparently a good marker of apoptosis, and detects early apoptotic changes in cells. However, not all cells that stain AV +ve are apoptotic, as AV will also bind to phosphotidylserine on the disrupted cell membrane of necrotic cells. Therefore, it is probably best to use a second 'marker' (see answer to question 3!). 2) My understanding is that there is nothing that is 'specific' for apoptosis. some markers are better than others. There are so-called 'gold standards', for example, DNA electrophoresis and electron microscopy. However, these may not be so practical. >From my point of view, I look at cells in sputum and BAL, so one cannot really apply these methods so easily. The key seems to be that one should always use more than one marker for apoptosis when looking at the phenomenon. For me, I use AV and propidium iodide (PI) along with light microscopy morphology (probably not the best method, but the only one easily available to us). 3) Yes, PI helps differentiate necrotic from apoptotic cells. Cells which are AV+PI- are held to be in the earlier stages of apoptosis. Cells which are AV+PI- are held to be in later apoptosis/necrosis. As for cells that are AV-PI+...well, I think that you will find a variety of opinions on this! This was one of the points that I raised in Feb. We have been looking at the granulocyte population in sputum/BAL, and have seen cells (though not many) that are AV-PI+. We feel that these may represent cells which are in an advanced state of necrosis. I hope that this is of some help. I hope that I am not adding to your confusion! Best of luck with your work. Martin Kelly On Wed, 10 May 2000 11:15:25 CEST Jose Benito <jbenito1@hotmail.com> wrote: > > Hi everyone. > I want to study apoptosis in PBMCs using the staining with annexin as a > marker of apoptotic cells. I have some questions regarding this issue: > 1.- How good the annexin staining correlates with apoptosis. Can a cell be > defined as apoptoic simply based in the annexin criterion? > 2.- Is there any test rally specific for the fenomenon of apoptosis? > 3.- What is the rationale of using propidium iodide in combination with > annexin? Is it to differentiate apoptotic from necrotic cells? If this is > the case, does it mean that cells positive for annexin and negative for PI > should be classified as apoptotic and double positive cells as necrotic? > Could you find cells positive for PI and negative for annexin? > Sorry for so many questions and Thanks > > José Miguel benito > Infectious Diseases Division > Hospital Carlos III > Madrid > ________________________________________________________________________ > Get Your Private, Free E-mail from MSN Hotmail at http://www.hotmail.com > ---------------------- Martin Kelly Department of Clinical Biochemistry, Institute of Clinical Science, Grosvenor Road, BELFAST. BT12 6BJ Tel No: +44 (0)28 9026 3267
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