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 *
****************************************************************************