Hi Lindsay, We have been performing annexin assays on adherent cells (MDCK, dog kidney cells) with a modicum of success. There are several ways that you can go about it and you may have to experiment a bit with the conditions. Obviously you have to get the cells off the flask if you want to use flow so they will need either trypsin (c0.5mg/ml), EDTA (c0.2mg/ml), a combination of both or scraping. We normally do the Annexin staining before detaching the cells. It is also important to save any cells that are in the supernatant and stain them as well - obviously this can be done in supension. The profiles that we see with adherent cells are never quite as clean as with suspension cells. There are altenatives, however. You could harvest and ethanol fix the cells and look for a sub-G1 peak although this is generally a much later event than PS exposure so may not be as useful. Looking for strand breaks is also an option but again may be a later event. If you have access to a Laser Scanning Cytometer looking for Annexin-postive cells is an ideal application for it as it can be done on the cells on the slide (but you will lose the suspension cells). Recently we have started using PhiPhiLux which is a caspase-3 substrate. In cells where caspase-3 is active, it cleaves the peptide to give a rhodamine-based fluorescent product. This seems to be less dependent on the method of cell harvesting and, in our hands, does give good separation of live, dead and apoptotic cells. There is info about this at: http://www.phiphilux.com/index.htm Hope that helps! Good luck! Derek On Fri, 25 Jun 1999, Lindsay Robinson wrote: > I have a question about the Annexin V apoptosis assay. We have been doing > this assay in our lab using lymphocytes and it has been working quite well. > Now we are trying to do the same assay using an adherent breast cancer cell > line (MDA-MB-231) and we are having some difficulties. Has anyone ever done > the Annexin V assay using adherent cells? We are not sure if we should be > doing the assay while the cells are still adhered to the plate or if we > should harvest the cells prior to adding the Annexin V. Also, we cannot > induce apoptosis in our adherent cells using our usual method (these tumor > cells are Fas resistant) and we have not been able to find a method for > doing this. Does anyone have any suggestions? Perhaps the Annexin V assay > is not the best option for measuring apoptosis of adherent cells? *************************************************************************** * Derek Davies Voice: (44) 0171 269 3394 * * FACS Laboratory, FAX: (44) 0171 269 3100 * * Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk * * London, UK * * * * Web Page: http://www.icnet.uk/axp/facs/davies/index.html * * * * In tenebris lux * ****************************************************************************
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