From: Maryann Demaria (maryann.demaria@es.nemc.org)
Date: Sun Mar 19 1995 - 15:36:10 EST
To clarify the LDS-751 question: 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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