title: Stability of cytokines in supernatants of stimulated mouse immune cells.
abstract: 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.
authors.affiliation: Department of Medical Pharmacology, School of Medicine, Akdeniz University, Dumlupınar Street, Kampus, 07070, Antalya, Turkey.
authors.forename: Ozbey
authors.initials: O
authors.lastname: G
authors.affiliation: The Toronto Hospital, University Health Network, Toronto, Canada; 2-805, MaRS Tower, 101 College Street, University Health Network and The Toronto Hospital, Toronto, Ontario, Canada M5G1L7.
authors.forename: Gorczynski
authors.initials: G
authors.lastname: R
authors.affiliation: Department of Medical Pharmacology, School of Medicine, Akdeniz University, Dumlupınar Street, Kampus, 07070, Antalya, Turkey.
authors.forename: Erin
authors.initials: E
authors.lastname: N
mesh_terms: D000818:Animals; D054426:Chemokine CXCL2 / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D003208:Concanavalin A / Q000494:pharmacology; D005260:Female; D005615:Freezing; D007371:Interferon-gamma / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D007378:Interleukins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008198:Lymph Nodes / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D008214:Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D055550:Protein Stability; D012034:Refrigeration; D013154:Spleen / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism
chemical_list: D054426:Chemokine CXCL2; C404915:Cxcl2 protein, mouse; D007378:Interleukins; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D003208:Concanavalin A; D007371:Interferon-gamma
keywords: ELISA; cytokines; secrotomes; stability