Cell fixation after Annexin V labelling ?

Derek Davies derek.davies at cancer.org.uk
Thu Feb 12 08:29:53 EST 2004


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


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