I'll be starting an experiment that requires to me immunize 125 mice
and do T cell proliferation and cytokine assays on splenic T cells
from all of them. I can't pool the groups. I've done all of the above
in the past, but never with so many samples. I expect the nightmare
in this experiment will be purification of T cells put into the assays. Does
anyone have suggestions for the most efficient method for isolating
T cells from 125 populations? I can space them over a three day
period since I have three main groups (three antigens) but I don't
want to subdivide those groups because I'm comparing different vaccination
methods and adjuvants for each of the three. I'm on a deadline schedule
that won't let me try each antigen in separate, more managable experiments.
Also, the company I'm at doesn't yet have a radiation license. I will
need to use a non-radioactive method for proliferation assays. I'm planning
to use a formazan based detection method but I'm amenable to changing
that. I've only used radioactive methods in the past and my impression, at
least
based on literature with associated kits, is that contaminating non-T cells can
add
a lot of noise to the assays. In my mind, panning might be a good
high-throughput
approach to isolate CD4+ cells out but it's not as clean as other methods.
Should
I expect this to be a huge issue for me with the formazan-based detection? Also,
if I use a pan-T isolation method, will CD8 cell contamination be a problem in
these assays?
Thanks in advance,
Stacy
