I have some experience of measurinf cell proliferation with Alamar blue. Is is really easy: 20 ul /W overnight and then read the plate with a spectrophotometer or a fluorescent plate reader. It is not really good anyway for really small proliferation (need at least something as 10 000 cpm...)
No idea for the other points, sorry.
sigrid
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
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