Hi Steve, 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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