Re: Questions about PBMCs, apoptosis induction, and flow cytometry analysis

Hi All,

I have a few of questions about performing apoptosis studies on PBMCs
and then analyzing by flow cytometry.  

1.	During the induction of apoptosis (camptothecin for 4 hours at
37oC, 5% CO2) approximately 50% of the cells attach to the surface of
the 6 well plates.  I use low attachment plates (Corning Costar(r) 6
Well Clear Flat Bottom Ultra Low Attachment Microplates, Product #3471),
and the PBMCs still adhere to the well surface.  When the plates are
continuously rocked during the incubation stage ~30% of the cells adhere
to the cell surface.  Does anyone know how to either stop the
attachment? If a different type of plate should be used? Or how to
reduce the percentage loss?  

2.	When analyzing PBMCs on the flow cytometer (the PBMCs have been
frozen in liquid nitrogen and then quickly thawed, and induced for
apoptosis for 4 hours with camptothecin as described above), has anyone
come across two populations of live PBMCs?  One of the populations has a
reduced forward scatter compared to the other population, but the side
scatter values are the same.  What is the cause of this?

3.	Camptothecin dissolved in DMSO forms precipitates, is this
common? And is there anyway to stop the precipitation? Can it be
dissolved in another reagent other than DMSO?

4.	Does anyone have experience with inducing apoptosis in PBMCs?
If so, which inducing agent do you use and for how long?  And what is
your maximum induction rate in the PBMC population?

Many thanks in advance for any help with any of these problems,

Dr Mark Hollier.
Centers for Disease Control and Prevention,
Atlanta, GA, USA
etu4@cdc.gov