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>Subject: EMA Staining Protocol
> I have been working with 5 uM ethidium monoazide (EMA) trying to identify
>nonviable cells in a 1% PFA fixed population of cells stained with fitc
>and pe conjugated antibodies. Problems I've had are dramatic changes in
>light scatter due to either the EMA or the DMSO used to solubilize the
>EMA, as well as a gross underestimate of nonviability in comparison to
>the same cells unfixed and PI stained. Additionally, the EMA induces a
>high fluorescent background. Anyone have any suggestions as to what stock
>concentration to make the EMA up in and how to solubilize it? Do people
>out there use EMA for this application of is there a better stain? Thanks
>in advance.
>Mark Rehse Rehsema@cellpro.cellpro.com
We have also been using EMA to identify dead cells post-fixation.
Using a range of 1-10 ug/ml (2.4-24 uM) of EMA( made up in isotonic pbs)
we found that EMA gave us the same percentage viablity after fix as
did PI pre-fixation;however the staining was only stable for a short
time (1-4 hours)- after 24 hours,stain had leaked out .
We observed no effect on cellular autofluoresence,but the FSC was
reduced after EMA treatment(we used a BD facscan for analysis).
We have similar questions to Mark i.e. has anyone out there tried
this technique and does anyone use it routinely? What problems
have other people encountered?
Any advice gratefully received.
Thanks in advance
Clare Hughes
CL_HUGHES@icrf.icnet.uk
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