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We are investigating Ca fluxes in PBL's from a variety of autoimmune diseases
and have found in the past using fura-2 loaded cells in a spectrofluorimeter
that activation via CD2 (using PHA-P) and CD3 (using antibody) are in some
cases reduced.
We have been trying to extend this data to look at subsets of T cells using
indo-1 loaded cells and a Coulter Epics Elite with UV laser. We can load the
cells OK with indo-1 and get very nice responses to ionomycin and also to
anti-CD3, but when we use PHA-P, which is supposed to act via the alternative
CD2 pathway we get very poor responses, if we get anything at all. We have
also tried using OKT11 (a CD2 antibody) together with a second anti-CD2,
conditions reputed to activate cells and these do not appear to induce a
Ca flux - even when these ae also crosslinked with a third antibody.
We wish to compare the two pathways to discover if they are equally affected
in disease. Does anyone have any experience of measuring CD2-induced Ca fluxes
and if so is there something I am doing wrong?
I'd appreciate any help you can give.
Steve
Dr SP Young
Dept of Rheumatology
University of Birmingham, UK
Email to s.p.young@bham.ac.uk Voice: 021-414-6780
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