Functional T-responders and T-regulators by cell sort?

Hi All,

I would like to post this question on behalf of one of the Researchers at
Rockefeller University for whom we sort cells. Sorts are done on BD FACSAria
with 70 um nozzle; pressure - 34 psi; frequency - 57 kHz; buffer - PBS
without Ca/Mg.	

Here is the question from Kamruz:
I'm trying to set-up a MLR with T-responders and T-regulators and am
experiencing tremendous difficulties to get flow-sorted T- responders
(CD3+,4+,25-,127+) to proliferate in response to allogeneic mature DCs (T
cell : DC ratio 10:1) or CD3/28 beads (T cell : bead ratio 1:3). I did the
same MLR with rosette-sep'd T responders (CD3+4+) and they proliferated very
strongly in response to the above stimuli. Therefore, I think flow-sorting
with CD3,4 and 127 antibodies binding to my T-responders might deliver some
type of signal to the cells that renders them non-responsive to TCR
signaling and co-stimulation and they don't proliferate. 

Is there anyone who is experiencing the same problem or anyone who can throw
his/her two cents in?

Thanks a lot,
Kamruz Darabi MD
Laboratory for Investigative Dermatology The Rockefeller University

Please feel free to reply to Kamruz (kdarabi@mail.rockefeller.edu); myself
(mazels@rockefeller.edu) or to the list.

Thanks,
Svetlana
*****
Svetlana Mazel, PhD
Flow Cytometry Resource Center
Rockefeller University