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I have done quite a bit of viable cell sorting using Hoechst 33342. It
has the advantage of being a very easy protocol - simply add Hoechst to
the cells at 37C and run them! Actually there are of course potential
pitfalls. The concentration of Hoechst needed to get a decent DNA profile
will vary from cell type to cell type - but is usually in the range
5-20ug/ml. Also the time of staining can vary, although 30-60 minutes
normally suffices.
Once on the sorter, I too was using dual beam set up, primary laser at
488nm (for scatter signals and also PI positive cells to gate out the
dead ones) and the secondary at UV (Argon ion, 351-363nm) to excite the
Hoechst. Hoechst fluorescence was collected between 390 and 500nm.
You have to be a bit careful if you are going to sort the cells for
subsequent culture as high levels of Hoechst are cytotoxic. Most of my
sorting has been done for subsequent protein or RNA analysis so a good
profile is the priority. If survival is paramount you may have to put up
with a sub-optimal DNA profile.
Hope that this is of some help
Derek
Derek Davies
FACS Laboratory
Imperial Cancer Research Fund
London, UK
http://www.icnet.uk/axp/facs/davies/index.html
** Insert song lyric here **
On Thu, 10 Oct 1996 jsfaust@wista.wistar.upenn.edu wrote:
>
> I have a user who would like to sort for live cells based on position in
> cell cycle. Does anyone have experience, a staining protocol, filter setup,
> hints, or tricks for using Hoechst for this purpose. I will be using a
> Coulter Elite, separated dual beam alignment, and a HeCd for excitation of
> the Hoechst. Thanks.
> Jeffrey S. Faust
> Philadelphia, PA
> 215-898-3811 (work)
>
>
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