Derek Sounds like doublets. As you note the FACScan/Calibur is not good at resolving doublets using H vs A. You need to use A vs W. If B-D had designed the instrument right you wouldn't have been forced to use A vs H with the dual laser system. If you can do calculated parameters on CellQuest (I don't use their software so I don't know if this is supported) you might try doing a partial square on the area parameter to try to mathematically pull them away from the diagonal. Larry Arnold Univ. of Nroth Carolina At 02:39 PM 8/5/97 +0100, you wrote: > > >Hello all, > >I hope that this is not a naive question, but hey, what the hell! > >I have a user who wishes to use PI to look at the cell cycle of thymocytes >which are surface stained with a direct conjugated CD4-FITC and another >biotinylated antibody detected with SA-APC. We have found that the best >way of preserving the antigen staining and getting a good PI DNA profile >is to use a modified fix and perm procedure. > >When we look at the DNA profile of unstained cells and FITC alone stained >cells, it is pretty good in terms of CV etc. However, in dual stained >specimens we see a build up in G2. Now I am presuming that this is due to >doublets. I am using a dual laser FACSCalibur, so I am forced to try to >use FL2-H and FL2-A to identify doublets. When we turn off the second >laser and look at FL2-A v FL2-W, we see a population that has the FL2-A of >a G2 cell but slightly more FL2-W signal, but this proves almost >impossible to separate on the FL2-H v FL2-A dot plot. > >By switching the staining so we use a SA-FITC, we see the same thing, so I >am presuming that the cross linking fixative is affecting the streptavidin >and causing cells to stick together. >Has anyone else seen this? >Is it a common effect? >What can I do about it? >I know I could use Hoechst, but I would rather keep it on a benchtop >machine if possible and the user wants to keep the biotinylated antibody >in there rather than get a whole series of direct conjugates. > >Thanks in advance for any help anyone can offer!! > >Derek > >**************************************************************************** >* Derek Davies Voice: (44) 0171 269 3394 * >* FACS Laboratory, FAX: (44) 0171 269 3100 * >* Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk * >* London, UK * >* * >* Web Page: http://www.icnet.uk/axp/facs/davies/index.html * >**************************************************************************** > > >
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