I am making some experiments with PBMC stimulated with LPS, but I have some difficulties because the basal levels of the cells without stimulation produced near to 1431,5 pg/mL of IL-8 cytokine. I would be grateful if somebody can sent me more information about the number of cells per well used or if I should consider some other variable.
Thank you!
Dear Eli
IL-8 is rapidly upregulated in monocytes and macrophages. We found negligible IL-8 mRNA in freshly isolated cells which was clearly upregulated just by adhesion and culture. But no doubt: LPS will multiply this basal expression manifold!
We routinely culture the cells as suggested above (1 Mill/ml) with and without stimulation. works fine.
I agree, the separation procedure seems to stimulate your cells.. 1 million per ml is OK.
Thank you very much. I I will decrease the concentration of serum in the medium and I going to prove the same concentration of cells (1mill/mL).
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