Hi Albert, To get the maximum amount of information out of the annexin assay (and indeed many other assays fir parts of the apoptotic pathway), the sample is best analysed unfixed so that it is possible to add a dye to discriminate the cells that are dead or in late stage apoptosis and distinguish them from cells that are in early apoptosis. In the annexin assay, early apoptiotic cells would be annexin positive and (say) propidium iodide negative. If the cells are live though, you have to remember that apoptosis is an ongoing event in some of your cells and there is a continuum between live, apoptotic and then dead cells. So there will always be a kinetic element in these analyses. I suspect that the advice is less to do with loss of signal and more to do with ensuring that cells are analysed within a similar time frame. I try to keep annexin experiments to a manageable number of samples, incubate for a given time at room temperature (normally about 15 minutes) and then keep samples on ice till analysed. Fixing would be OK but you would lose the distinction between early and late apoptotic cells Good luck! Derek >Dear All, > >I have used the BD Annexin V apoptosis kit and had good success with >it. However, the protocol supplied by Pharmingen recommended >processing the labelled samples within a couple hours of labelling. >Has anyone tried fixing the labelled cells or put the labelled cells >on ice if one is processing large amount of samples without loss of >signal ? > >Your input is much appreciated. > >Albert Tai -- *************************************************************** Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 London Research Institute, e_mail: derek.davies@cancer.org.uk Cancer Research UK mobile: 07790 604112 44 Lincolns Inn Fields, London, UK. Web Page: http://sci.cancerresearchuk.org/axp/facs/davies/index.html In tenebris lux ***************************************************************Received on Thu Feb 12 11:38:00 2004
This archive was generated by hypermail 2.1.8 : Fri Feb 13 2004 - 03:12:02 EST
