Hi Robert, It is true that you have to be careful with harvesting the cells as it can be quite easy to induce membrane damage but it is usually possible to minimise this after a while! It is a good idea to do a time course as I have found that the time when cells are annexin +ve but PI-ve can be quite short and may well be over after 24 hours. You dont say if you see an increase in the percentage of dead cells after 24/48 hours? If so then that is a good indication that apoptosis has accurred but that the cells have passed through that stage to necrosis. Looking at earlier points may reveal the apoptotic population. As will all assays that rely on live/dead cell discrimination, the percentage of cells that are 'apoptotic' at any particular time point may be relatively small. It is also sometimes productive to do some other assay in parallel - even looking for sub-G1 cells may help - you can use the same cell as for the annexin assay, just fix them in ethanol after running. Good luck! Derek On Mon, 1 May 2000, Robert Connelly wrote: > We are having some problems developing an annexin V/PI assay for the > SK-OV-3 and BG-1 both epithelial ovarian cancer cell lines. We exposing > the cells in vitro to cisplatin for 24, and 48 hours. We are using the > Clontech EGFP-annexin V kit with PI. > The literature is controversial and confusing particularly in the > harvesting of the cells from the substrate. We have been harvesting with > Trypsin-EDTA and scraping. Unfortunately scraping increases ( compared > to the Trypsin-EDTA group) the annexin negative/ PI positive population > in the untreated control group. > To date we have not seen any annexin V positive results after 24 or 48 > hours of cisplatin exposure. Our next experiments > we be a time course study of cisplatin exposure for PS externalization > to occur. > If the group could shed some light or give constructive suggestions it > would be greatly appreciated. ************************************************************************ 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 *************************************************************************
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