Hi Stacy!
Be aware that large experiments never turn out to be as good as the small ones... Leaving T cells on ice until you have purified the cells from all your mice will inevitable decrease their health-status... So make sure you don't start with one group and end with another... Proliferation counts always turn out to be a lot lower in big experiments, so make sure you get all the help you can get, and be quick! I would also recommend R&D columns, maybe even the CD3+, if you don't need pure CD4+ T-cells. They are a lot cheaper, and quicker to use. I don't think the CD8's would disturb your assay. Regarding measuring proliferation, have you considered mesuring IL-2 (supernatants, after 24 h, will decrease after that) in a simple ELISA. Worth to think about. I have bad experience with Alamar Blue... Seems to very insensitive.
One thing you should keep in mind for the next time is that you really need to optimize your experiments in advance. I don't know which APC you are using (I guess you are using something, since you want to use purified T cells), but the ratio between these to cell types is very important in order to get a good response, along with antigen concentration, of course, and time kinetics, if you are measuring proliferation in other ways than with IL-2. It is always a shame when there is not enough time to set up an assay before the real experiment is due... Usually a waste of time and money in my opinion.
/Anna-Karin
Stacy Ferguson wrote:
> 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
http://biowww.net/mynews/tree.php?group_name=bionet.immunology&begin=0
