European Cytokine Network

, Volume 25, Issue 2, pp 30–34 | Cite as

Stability of cytokines in supernatants of stimulated mouse immune cells

  • G. Ozbey
  • R. Gorczynski
  • N. Erin
Research Article


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°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-γ 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.

Key words

stability cytokines secrotomes ELISA 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Meager A. Measurement of cytokines by bioassays: theory and application. Methods 2006; 38: 237.PubMedCrossRefGoogle Scholar
  2. 2.
    Chu WM. Tumor necrosis factor. Cancer Lett 2013; 328: 222.PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Saha B, Jyothi Prasanna S, Chandrasekar B, Nandi D. Gene modulation and immunoregulatory roles of interferon gamma. Cytokine 2010; 50: 1.PubMedCrossRefGoogle Scholar
  4. 4.
    Ouyang W, Rutz S, Crellin NK, Valdez PA, Hymowitz SG. Regulation and functions of the IL-10 family of cytokines in inflammation and disease. Annu Rev Immunol 2011; 29: 71.PubMedCrossRefGoogle Scholar
  5. 5.
    Nishimoto N. Interleukin-6 as a therapeutic target in candidate inflammatory diseases. Clin Pharmacol Ther 2010; 87: 483.PubMedCrossRefGoogle Scholar
  6. 6.
    Zhu S, Qian Y. IL-17/IL-17 receptor system in autoimmune disease: mechanisms and therapeutic potential. Clin Sci (Lond) 2012; 122: 487.CrossRefGoogle Scholar
  7. 7.
    Acharyya S, Oskarsson T, Vanharanta S, Malladi S, Kim J, Morris PG, Manova-Todorova K, Leversha M, Hogg N, Seshan VE, Norton L, Brogi E, Massague J. A CXCL1 paracrine network links cancer chemoresistance and metastasis. Cell 2012; 150: 165.PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell 2010; 140: 883.PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Yamaguchi Y, Ohshita A, Kawabuchi Y, Ohta K, Shimizu K, Minami K, Hihara J, Miyahara E, Toge T. Adoptive immunotherapy of cancer using activated autologous lymphocytes-current status and new strategies. Hum Cell 2003; 16: 183.PubMedCrossRefGoogle Scholar
  10. 10.
    House RV. Cytokine measurement techniques for assessing hypersensitivity. Toxicology 2001; 158: 51.PubMedCrossRefGoogle Scholar
  11. 11.
    Keustermans GC, Hoeks SB, Meerding JM, Prakken BJ, de Jager W. Cytokine assays: an assessment of the preparation and treatment of blood and tissue samples. Methods 2013; 61: 10.PubMedCrossRefGoogle Scholar
  12. 12.
    De Jongh R, Vranken J, Vundelinckx G, Bosmans E, Maes M, Heylen R. The effects of anticoagulation and processing on assays of IL-6, sIL-6R, sIL-2R and soluble transferrin receptor. Cytokine 1997; 9: 696.PubMedCrossRefGoogle Scholar
  13. 13.
    Flower L, Ahuja RH, Humphries SE, Mohamed-Ali V. Effects of sample handling on the stability of interleukin 6, tumour necrosis factor-alpha and leptin. Cytokine 2000; 12: 1712.PubMedCrossRefGoogle Scholar
  14. 14.
    Kenis G, Teunissen C, De Jongh R, Bosmans E, Steinbusch H, Maes M. Stability of interleukin 6, soluble interleukin 6 receptor, interleukin 10 and CC16 in human serum. Cytokine 2002; 19: 228.PubMedCrossRefGoogle Scholar
  15. 15.
    Erin E, Kale S, Tanriover G, Koksoy S, Duymus O, Korcum AF. Differential characteristics of heart, liver, and brain metastatic subsets of murine breast carcinoma. Breast Cancer Res Treat 2013; 139: 677.PubMedCrossRefGoogle Scholar
  16. 16.
    Moehrlen U, Lechner A, Baumel M, Dostert K, Rohrl J, Meuli M, Mannel DN, Hamacher J. Immune cell populations and cytokine production in spleen and mesenteric lymph nodes after laparoscopic surgery versus conventional laparotomy in mice. Pediatr Surg Int 2012; 28: 507.PubMedCrossRefGoogle Scholar
  17. 17.
    Al-Mariri A, Tibor A, Mertens P, De Bolle X, Michel P, Godfroid J, Walravens K, Letesson JJ. Induction of immune response in BALB/c mice with aDNAvaccine encoding bacterioferritin or P39 of Brucella spp. Infect Immun 2001; 69: 6264.PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    de Jager W, Bourcier K, Rijkers GT, Prakken BJ, Seyfert-Margolis V. Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays. BMC Immunol 2009; 28: 10.Google Scholar
  19. 19.
    Friebe A, Volk HD. Stability of tumor necrosis factor alpha, interleukin 6, and interleukin 8 in blood samples of patients with systemic immune activation. Arch Pathol Lab Med 2008; 132: 1802.PubMedGoogle Scholar
  20. 20.
    Erin N, Ulusoy O. Differentiation of neuronal from non-neuronal Substance P. Regul Pept 2009; 152: 108.PubMedCrossRefGoogle Scholar

Copyright information

© John Libbey Eurotext 2014

Authors and Affiliations

  1. 1.Department of Medical Pharmacology, School of MedicineAkdeniz University, Dumlupınar Street, KampusAntalyaTurkey
  2. 2.The Toronto HospitalUniversity Health NetworkTorontoCanada
  3. 3.University Health Network and The Toronto HospitalTorontoCanada

Personalised recommendations