Re: fluorescent nuclear stain

<< Previous Message | Next Message >>
From:"R.Wadley" <> (by way of histonet)
To:histonet <>
Content-Type:text/plain; charset="us-ascii"

	Dear Judy,

	PI is a nuclear marker, we use it for flow cytometry, laser scanning
cytometry & confocal work.  However, it will stain RNA, so the addition of
some RNase to your PI stain should fix that problem.  Normally the RNA
staining should be green not red, perhaps your PI concentration is too
high?  The other problem is that PI does not bind tightly to DNA & will
leach out of your cells over time.  Normally we add PI & begin our analysis
without washing.  Times for PI staining vary from instant to about 10
minutes depending on the instrument & the sample.
	Working with GFP can be a right pain.  Depending on whether its
bound or
not GFP will diffuse out of cells permiablised to accept a nuclear stain.
Fixing the cells is usually bad news for GFP fluorescnece.  Although there
was some information on the Cytometry mailing list recently about a method
that fixed cells & maintained the fluorescence properties of BOUND GFP.
	Depending on the instrument we are using we have used DAPI,
Hoescht, PE &
PI to stain nuclei.  DAPI & Hoescht are both in the blue so no good to you,
PE is an orange dye so you have to do compensation to try & sort the
signals out.
	The only other thing I can think of is that somehow your treatment
of the
cells has allowed DNA into the cytoplasm?  Surely not.

	Hope this helps


	Rob W.

At 04:16 PM 3/5/99 -0500, you wrote:
>We are trying to localize gfp expression in transfected HeLa cells and
>would like to use DNA counterstain to distinguish between nuclear and
>cytoplasmic distribution. We have tried propidium iodide which I
>thought is a nuclear marker, however stained the entire cell. We have
>also tried Yo-Pro-3, from Molecular Probes, and it, too, labelled
>both the nucleus and the cytoplasm. Our confocal microscope laser does
>not have excitation for DAPI.
>Any suggestions for another 'red' DNA marker would be highly appreciated.

R. Wadley, B.App.Sc, M.L.S
Laboratory Manager
Cellular Analysis Facility
School of Microbiology & Immunology
UNSW, New South Wales, Australia, 2052
Ph (BH) 	+61 (2) 9385 3517
Ph (AH)	+61 (2) 9555 1239
Fax 	+61 (2) 9385 1591
	(Under development)

<< Previous Message | Next Message >>