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Can't activate C57Bl/6 CD4+ T cells!

T cell CD4 th1

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#1 LittleMissTcell



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Posted 30 November 2011 - 03:51 AM

Hi everyone

I'm trying to polarise CD4+ T cells towards Th1 and having really limited success. I've actually been working with these cells for a year now (the whole first year of my PhD) and they activated fine up until about 6 months ago. The strange thing is that I can activate the cells if I culture whole spleen but if I purify them first they don't activate at all.

My protocol is:
CD4+ T cells purified by miltenyi or stemcell magnetic beads. Cultured at 1x106 cells/ml for 48-72 hours with 1ug/ml antiCD3 and antiCD28 + 10ng/ml IL-12. I usually use RPMI + 10% FBS though recently I've been trying serum free x-vivo media though getting even less survival/activation.

Has anyone got any ideas what might be going wrong or what I could change to try and fix it? It is the most frustrating thing!


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