Hello,
I purify CD3+ T Lymphocytes from mouse spleens and lymph nodes, then immediately activate them with CD3/CD28 beads and 20U/ml Il-2.
At first, the cells proliferate and viability is high, but after 5-6 days they stop proliferating and start dying. Adding more Il-2 or CD3/CD28 beads doesn't seem to help. Is there any way to keep my lymphocytes proliferating in culture longer?
Thanks for your help.
The same question was recently asked and my answer was as below:
You might find some work by Carl June in UPenn helpful. Immobilized MHC/peptide tetramer plus anti CD28 and conditioned media would stimulate the CD4 cells to proliferate in vitro. Take a look at this publication: http://www.ncbi.nlm.nih.gov/pubmed/17375070
You could try reducing the concentration of IL-2 to 5-10 U/ml and also try adding some IL-7 and IL-15, which should help to keep the T cells alive. Additionally, The T cells need to be harvested and cultured in fresh media (containing cytokines) every 3-4 days (cycle). CD3/CD28 beads should be added to culture every alternate cycle, since strong/continuous TCR signaling can also cause cell death.
My guess is that after 3-4 days, your media starts turning yellow. This is an indication that your culture conditions are no longer optimal: too many cells for not enough food.
- adding more stimuli will only kill your cells faster. IL-2 just helps getting the cells to proliferate faster.
- try removing 50% of your media and replacing it with fresh media. You could also decrease the number of CD3/CD28 beads you started with by half and decreasing the amounts of IL-2 by 90% or completly.
Dear Jean-David, used concentration of IL-2 seems too high. At this concentration, formation of LAK cells is possible. Decrease it to 2-5 IU/ml. Good luck!
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