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Briefly, the project is isolating fetal nucleated red blood cells from
maternal peripheral blood and then performing FISH analysis on the sorted
cells. Non-nucleated red cells cause problems so a nuclear dye is always
incorporated in our sorts. On the Vantage, I use Hoechst 33342 in combination
with other fluorochromes. I use Hoechst in all our staining protocols: both
unfixed and fixed cells. We are trying to find a viable nuclear dye to be
used on our FacStar Plus that only has a 488 laser and LDS-751 was the only
viable dye I could think of. Fixed samples aren't really a problem. However,
the red nuclear dyes do bother our FISH people because they are so bright and
they are using the same fluochromes for their FISH probes. Hoechst is nice
because it really doesn't bother the FISH people.
The problems that we have with LDS-751 so far have been that it does not
discriminate between nucleated and non-nucleated cells as well as Hoechst
does, even non-nucleated cells take up the dye and we have had trouble with
time course experiments. We have trouble repeating the staining pattern for a
given cell population even while keeping everything constant.
Suggestions on how to use the dye, info on how it binds to DNA, and
suggestions on other viable DNA binding dyes would be appreciated.
I would appreciate any suggestions and want to thank the people that have
already answered.
Thanks
MaryAnn DeMaria
New England Medical Center
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