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Flow Cytometry Protocols pp 165–178Cite as

Multiparameter Intracellular Cytokine Staining

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 699))

Abstract

Intracellular cytokine staining (ICS) is a popular method for visualizing cellular responses, most often T-cell responses to antigenic or mitogenic stimulation. It can be coupled with staining for other functional markers, such as upregulation of CD107 or CD154, as well as phenotypic markers that define specific cellular subsets, e.g. effector and memory T-cell compartments. Recent advances in multicolor flow cytometry instrumentation and software have allowed the routine combination of 8–12 (or more) markers in combination, creating technical and analytical challenges along the way, and exposing a need for standardization in the field. Here, we will review best practices for antibody panel design and procedural variables for multicolor ICS, and present an optimized protocol with variations designed for use with specific markers and sample types.

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References

  1. Jung, T., Schauer, U., Heusser, C., Neumann, C., and Rieger, C. (1993) Detection of intracellular cytokines by flow cytometry, J Immunol Methods 159, 197–207.

    Article  CAS  Google Scholar 

  2. Prussin, C. and Metcalfe, D. D. (1995) Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies, J Immunol Methods 188, 117–128.

    Article  CAS  Google Scholar 

  3. Waldrop, S. L., Pitcher, C. J., Peterson, D. M., Maino, V. C., and Picker, L. J. (1997) Determination of antigen-specific memory/effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency, J Clin Invest 99, 1739–1750.

    Article  CAS  Google Scholar 

  4. Suni, M. A., Picker, L. J., and Maino, V. C. (1998) Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry, J Immunol Methods 212, 89–98.

    Article  CAS  Google Scholar 

  5. Maecker, H. T., Dunn, H. S., Suni, M. A., Khatamzas, E., Pitcher, C. J., Bunde, T., Persaud, N., Trigona, W., Fu, T. M., Sinclair, E., Bredt, B. M., McCune, J. M., Maino, V. C., Kern, F., and Picker, L. J. (2001) Use of overlapping peptide mixtures as antigens for cytokine flow cytometry, J Immunol Methods 255, 27–40.

    Article  CAS  Google Scholar 

  6. Maecker, H. T. (2009) Multiparameter flow cytometry monitoring of T cell responses, Methods Mol Biol 485, 375–391.

    Article  CAS  Google Scholar 

  7. Maecker, H. T., Frey, T., Nomura, L. E., and Trotter, J. (2004) Selecting fluorochrome conjugates for maximum sensitivity, Cytometry A 62, 169–173.

    Article  Google Scholar 

  8. Maecker, H. T., Rinfret, A., D’Souza, P., Darden, J., Roig, E., Landry, C., Hayes, P., Birungi, J., Anzala, O., Garcia, M., Harari, A., Frank, I., Baydo, R., Baker, M., Holbrook, J., Ottinger, J., Lamoreaux, L., Epling, C. L., Sinclair, E., Suni, M. A., Punt, K., Calarota, S., El-Bahi, S., Alter, G., Maila, H., Kuta, E., Cox, J., Gray, C., Altfeld, M., Nougarede, N., Boyer, J., Tussey, L., Tobery, T., Bredt, B., Roederer, M., Koup, R., Maino, V. C., Weinhold, K., Pantaleo, G., Gilmour, J., Horton, H., and Sekaly, R. P. (2005) Standardization of cytokine flow cytometry assays, BMC Immunol 6, 13.

    Article  Google Scholar 

  9. Suni, M. A., Dunn, H. S., Orr, P. L., deLaat, R., Sinclair, E., Ghanekar, S. A., Bredt, B. M., Dunne, J. F., Maino, V. C., and Maecker, H. T. (2003) Performance of plate-based cytokine flow cytometry with automated data analysis, BMC Immunol 4, 9.

    Article  Google Scholar 

  10. Disis, M. L., Dela Rosa, C., Goodell, V., Kuan, L. Y., Chang, J. C., Kuus-Reichel, K., Clay, T. M., Kim Lyerly, H., Bhatia, S., Ghanekar, S. A., Maino, V. C., and Maecker, H. T. (2006) Maximizing the retention of antigen specific lymphocyte function after cryopreservation, J Immunol Methods 308, 13–18.

    Article  CAS  Google Scholar 

  11. Waldrop, S. L., Davis, K. A., Maino, V. C., and Picker, L. J. (1998) Normal human CD4+ memory T cells display broad heterogeneity in their activation threshold for cytokine synthesis, J Immunol 161, 5284–5295.

    CAS  Google Scholar 

  12. Nomura, L. E., Walker, J. M., and Maecker, H. T. (2000) Optimization of whole blood antigen-specific cytokine assays for CD4(+) T cells, Cytometry 40, 60–68.

    Article  CAS  Google Scholar 

  13. Maecker, H. T., Ghanekar, S. A., Suni, M. A., He, X. S., Picker, L. J., and Maino, V. C. (2001) Factors affecting the efficiency of CD8+ T cell cross-priming with exogenous antigens, J Immunol 166, 7268–7275.

    CAS  Google Scholar 

  14. Nomura, L. E., DeHaro, E. D., Martin, L. N., and Maecker, H. T. (2003) Optimal preparation of rhesus macaque blood for cytokine flow cytometric analysis, Cytometry 53A, 28–38.

    Article  Google Scholar 

  15. Nomura, L. E., Emu, B., Hoh, R., Haaland, P., Deeks, S. G., Martin, J. N., McCune, J. M., Nixon, D. F., and Maecker, H. T. (2006) IL-2 production correlates with effector cell differentiation in HIV-specific CD8+ T cells, AIDS Res Ther 3, 18.

    Article  Google Scholar 

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Acknowledgments

Details of this protocol were optimized by Laurel Nomura and Maria Suni (BD Biosciences).

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Authors and Affiliations

  1. Institute for Immunity, Transplantation, and Infection, Stanford University, Stanford, CA, USA

    Patricia Lovelace & Holden T. Maecker

Authors
  1. Patricia Lovelace
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  2. Holden T. Maecker
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Corresponding author

Correspondence to Holden T. Maecker .

Editor information

Editors and Affiliations

  1. Medical Center, Flow Cytometry Core Facility, George Washington University, I Street NW 2300, Washington, 20037, District of Columbia, USA

    Teresa S. Hawley

  2. Medical Center, Dept. Anatomy & Regenerative Biology, George Washington University, I Street NW 2300, Washington, 20037, District of Columbia, USA

    Robert G. Hawley

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© 2011 Springer Science+Business Media, LLC

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Lovelace, P., Maecker, H.T. (2011). Multiparameter Intracellular Cytokine Staining. In: Hawley, T., Hawley, R. (eds) Flow Cytometry Protocols. Methods in Molecular Biology, vol 699. Humana Press. https://doi.org/10.1007/978-1-61737-950-5_8

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  • DOI: https://doi.org/10.1007/978-1-61737-950-5_8

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61737-949-9

  • Online ISBN: 978-1-61737-950-5

  • eBook Packages: Springer Protocols

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