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There are several third colour reagents that you could use, but most of
our users here use either streptavidin-TriColor (Caltag, cat no SA1006) or
streptavidin-CyChrome (Pharmingen, cat no 13038A). In our hands these
agive good signals.
As for DNA stains, as you have a UV laser your best bet is to use one of
the Hoechst dyes. If your cells are live Hoechst 33342 will do the job
(You may need to play around with the concentration, but 10ug/ml is a good
starting point). This is the approach that we have used to assess the cell
cycle status of sub-populations of triple stained thymocytes.
It may also be possible to use To-Pro-3 as the DNA stain, using excitation
by the HeNe laser, although I think that if you are using relatively high
power on the 488 laser, you would see some excitation in the third
channel, so it is probably of limited use, also the Hoechst DNA profiles
are generally nicer.
Hope this helps,
Derek
Derek Davies
FACS Laboratory,
Imperial Cancer Research Fund,
London, UK
http://www.icnet.uk/axp/facs/davies/index.html
On Wed, 8 Jan 1997, Steve
Neben wrote:
>
> Dear Flow-ers:
>
> I'm looking for a realiable method of staining bone marrow cells for
> simultaneous cell cycle analysis and 3-color surface
> immunofluorescence. Antibodies are FITC-, PE-, and biotin-conjugated.
> I'm using a FACStarPlus with Argon, He-Ne, and UV lasers. Can anyone
> suggest a good DNA stain and avidin conjugate that will work for this
> configuration ?
>
> Steve Neben
> Genetics Institute
> Cambridge, MA
>
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
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If you have any comments please direct them to
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Phone: (765)-494-0757;
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