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 *
>****************************************************************************
>
>
>