Ca flux in frozen PBMC

I am trying to look at Ca flux in human peripheral T cells in response
to anti-CD3 stimulation using Indo-1 on a MoFLo. I have had very good
success with fresh cells. However, I have had very limited success with
frozen PBMC (frozen in LN2 in 90% FCS and 10% DMSO). The frozen cells
respond well to ionomycin stimulation (5ug/ml) but hardly respond to CD3
stimulation. Has anyone else attempted to measure Ca flux in frozen
cells? Any tips? We cannot avoid using frozen cells, so it'd be nice if
it is possible to measure the Ca flux in these cells.

Thanks

Matthew Henry

CD45 Group
Edward Jenner Institute for Vaccine Research
Newbury
UK 
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