I'm working on a Th17 push assay where I isolate CD4 T cells and culture 1.25 x 10^6 CD4s with αCD3 (2.5 μg/mL), αIL-4 (10 μg/mL), αIFNγ(10 μg/mL), IL-6 20 μg/mL), TGFB1 (5 ng/mL), and IL-23 (10 μg/mL), along with 6.25 x 10^6 irradiated feeder cells (the non-CD4 cells from the isolation step, irradiated at 3000 rad in R10 media) in 2.5 mL R10 media/well in a 12-well plate for 5 days, splitting wells on day 3.
I get pretty good IL-17 production (~25% of live CD4s) but they just aren't proliferating as much as my lab has seen previously. I get about 4 x 10^6 back. It may just be that I'm still pretty new at immunology research and my technique isn't great but I had a lab mate watch me and she said I did fine.
Any to increase final yield?
Dear Joy Shepard
Check these might be helpful and answer your Q:
T cells from irradiated animals have low proliferative capacity and cytokine production upon TCR-mediated stimulation.
Article Ionizing Radiation Impairs T Cell Activation by Affecting Me...
AND
https://www.jimmunol.org/content/jimmunol/192/7/3101.full.pdf
To be certain your T cells are not proliferating much you may be interested in using Cell Proliferation dyes like these from Thermofisher: https://www.thermofisher.com/us/en/home/life-science/cell-analysis/flow-cytometry/flow-cytometry-assays-reagents/cell-proliferation-flow-cytometry/improved-cfse-alternatives-cell-proliferation.html
This will also allow you to understand if all T cells are dividing very few times or if very few T cells are dividing many times, as the division spectrum will look qualitatively different.
Best,
Michael
It may be that your cell concentrations are too high. If I am reading correctly, it sounds like your starting concentration is 3million total cells/ml. That doesn't leave those CD4s much room to go. I would decrease the total cell concentration to about 1 million cells/ml and consider reducing your ratio of CD4 T cells to feeder cells. But if you are following an exact protocol from other lab members, then I'm not sure!
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