On Wed, 10 May 2000, Jose Benito wrote: > 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? It is generally *not* a good idea to base assessment of apoptosis on a single method. Most methods, certainly those used in flow, are based on the detection of a certain part of the apoptotic pathway. It is likely that the rate of apoptosis and the relevant contributions of things such as caspase activation, cahnges in mitochondrial membrane potential, changes in the cell membrane and alteration to DNA will vary with cell type, method of apoptosis induction and time of measurement after induction. As you know, annexin binds to phosphatidylserine residues that are externalised in apoptosis (but is that the only time they are externalised?), so will give you an idea, but it is best to do at least one other method in parallel as well as using that old standby, the microscope. There is a paper in the latest "Cytometry" that shows DNA fragmentation preceeding annexin staining and I too have anecdotal evidence that supports this in some cell types. Looking for sub-G1 cells is a very quick and easy assessment. > 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? The rationale is that in early apoptotic cells ie annexin positive/ PI negative ones, the cells still retain the ability to exclude the DNA discriminatory dyes such as PI, 7AAD or TO-PRO-3. As the cell passes through apoptosis they become gradually more permeable. This can make the assessment of apoptosis a little subjective using this method. Obviously you need a control (untreated cells) to assess the background level and if you treat the cells with a known apoptotic inducer then it may be safe to assume that cells that appear in the double positive population have got there via an apoptotic pathway. It is also possible to see cells that are PI+ve but annexin -ve but _in general_ these are nuclei ie cells that have passed through necrosis and lost their cytoplasm (and hence their ps residues). Again, checking microscopically will help here. Hope this helps, Derek ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK mobile: 07790 604112 Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:19 EST