In article <369A72D2.50D4 at wpsmtp.siumed.edu>, kimxtyus at wpsmtp.siumed.edu wrote:
> I am interested in stimulating human lymphocytes with IL-2 and anti-CD3.
> Is this the best method, and if so at what concentrations. I will
> further stimulate with follicular fluid and IVF insemination media and
> measure Th2 cytokines in the supernatants. Any suggestions?
You might consider coating the cell culture plates you're going to use
with 2 microgr/ml of anti-CD3 in some sterile coating buffer (using .2
micrometer filtered bi-carboinate buffer), eg. the OKT3 monoclonal instead
of using it in solution. It's a fairly standard procedure these days,
thought (but not proven) to mimic antigen stimulation by an antigen
presenting cell. It might be a good idea to have a non-stimulated culture
going in parallel as a negative control.
If you're going to look for IL-4, I would suggest harvesting the
supernatant after 48H incubation, quickly freezing the conditioned medium
in small aliquots (say, .5 ml) and storing them at -70°C until you're
ready to analyze a lot of them together. ELISA is a good way of looking
for IL-4; protein level measurement (unlike RT-PCR), very good specificity
and fast&easy to perform (unlike bioassays). A lot of companies sell
matched pair antiIL-4 antibodies for ELISA, by the way, so if you're going
to do a lot of assays it might be financially interesting to have a look
at those. We have very good experience with Biosource antibodies.
Hope this helps,
Guy
--------Guy Hermans, PhD student------------
MS research Unit Tel 0032 (0) 11/26.92.07
Dr. L. Willems-Institute Fax 0032 (0) 11/26.92.09
Belgium
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