In article (Dans l'article) <EK8rMx.5pC at oci.utoronto.ca>, "Rizwan Haq"
<haqr at oci.utoronto.ca> wrote (écrivait) :
> We are interested in detecting BrdU incorporation in GFP+ cells as an assay
> of cell proliferation. The approach we want to take is to transiently
> transfect cells with gene of interest and GFP. After 48hrs, we label the
> cells in BrdU-containing media, and measure the number of cells that are
> both GFP+ and BrdU+ compared to GFP+ BrdU- cells (using immunofluorescence).
> This has been reported before (Biotechniques, 1997, p. 88), but the paper
> lacks details on how this was done. Specifically,
>> - how is the GFP preserved when labeling the BrdU?
>> - what fixation methods preserve the GFP fluoresence?
>> Does anyone have any advice regarding these? Thanks in advance to any help
> you can provide. Either post or email haqr at oci.utoronto.ca, and I'll
> summarize for the newsgroup.
> Rizwan
Clontech says that a paraformaldehyde fixation do preserve fluorescence. In
our lab, we use a short methanol fixation. It works, but GFP is somewhat
bleached. Make up your mind and good luck !
Yann
--
Yann ABRAHAM
Institut Curie
Laboratoire "Biologie du cycle cellulaire et de la Motilité"
UMR 144 - C.N.R.S.
26 rue d'Ulm
75231 Paris cedex 5
