Stability of cytokines in supernatants of stimulated mouse immune cells.

Measurements of cytokines in cell culture supernatants are widely used to evaluate the immune response. Cytokine levels in secretomes are usually quantified using enzyme-linked immunosorbent assays (ELISA), which have easy, sensitive, specific, rapid, cost-effective, and reproducible protocols. To our knowledge, the stability of cytokines in secretomes has not been hitherto investigated. We present data that involve; time-dependent changes during storage at +4°C, and the effects of freeze-thaw cycles in samples frozen at -80(o)C, instant freezing of samples with liquid nitrogen, and addition of protease inhibitors on the stability of certain cytokines (TNF-α, MIP-2, IFN-γ, IL-6, IL-10, IL-17A), in secrotomes of spleen and lymph nodes from tumor-bearing animals. Our results show that IL-6 remains stable, MIP-2, IFN-γ and IL-10 are somewhat stable, while TNF-α and IL-17A are degradable cytokines: instant freezing by liquid nitrogen or adding protease inhibitor does not preserve the stability of these cytokines. From these results it can be concluded that, if possible, TNF-α measurements should be perform in fresh samples, and IL-17A and IL-10 samples can be stored at -80°C, but should be used at the first thaw.