Abstract
Analysis of intracellular cytokines is extremely important in the clinical treatment of numerous diseases. Flow cytometry (FCM) is a highly effective technique that detects intracellular cytokines using specific fluorescence-labeled antibodies. The common steps of this assay include cell collection, fixation, permeabilization, blocking, intracellular staining and analysis by FCM. This technique also allows for analyzing the biological function of cytokines. In this chapter, we describe a modified method to detect the specific intracellular cytokine staining using FCM, with an emphasis on the effects of variables including samples, temperature, buffers, data acquisition, and analysis.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Shi Z, Yang WM, Chen L-P et al (2012) Enhanced chemosensitization in multidrug-resistant human breast cancer cells by inhibition of IL-6 and IL-8 production. Breast Cancer Res Treat 135:737–747
Suni MA, Picker LJ, Maino VC (1998) Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry. J Immunol Methods 212:89–98
Baraldi-Junkins CA, Beck AC, Rothstein G (2000) Hematopoiesis and cytokines: relevance to cancer and aging. Hematol Oncol Clin North Am 14:45–61
Fasciani A, D’Ambrogio G, Bocci G et al (2001) Vascular endothelial growth factor and interleukin-8 in ovarian cystic pathology. Fertil Steril 75:1218–1221
Bamberger E, Perrett C (2002) Angiogenesis in epithelian ovarian cancer. Mol Pathol 55:348–359
Duan Z, Feller AJ, Penson RT et al (1999) Discovery of differentially expressed genes associated with paclitaxel resistance using cDNA array technology: analysis of interleukin (IL) 6, IL-8, and monocyte chemotactic protein 1 in the paclitaxel-resistant phenotype. Clin Cancer Res 5:3445–3453
Thorpe R, Wadhwa M, Bird C et al (1992) Detection and measurement of cytokines. Blood Rev 6:133–148
Aggarwal B., Puri R. (1995) Common and uncommon features of cytokines and cytokine receptors: an overview. In: Human cytokines: their role in disease and therapy, vol 3. Blackwell Science, Cambridge
Jung T, Schauer U, Heusser C et al (1993) Detection of intracellular cytokines by flow cytometry. J Immunol Methods 159:197–207
Sander B, Andersson J, Andersson U (1991) Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol Rev 119:65–93
Schmid I, Uittenbogaart CH, Giorgi JV (1991) A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification. Cytometry 12:279–285
Foster B., Prussin C., Liu F, et al. (2007) Detection of intracellular cytokines by flow cytometry. Curr Protoc Immunol 6.24. 21–26.24. 21
Foster B., Prussin C. (2002) Detection of Intracellular Cytokines by Flow Cytometry. Curr Protoc Immunol
Prussin C, Metcalfe DD (1995) Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Methods 188:117–128
Andersson U, Andersson J (1994) Immunolabeling of cytokine-producing cells in tissues and in suspension. Cytokine Producing Cells 32–49
Sander B, Höidén I, Andersson U et al (1993) Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen: cytokine detection by immunoassay and intracellular immunostraining. J Immunol Methods 166:201–214
Lanier L, Warner N (1981) Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis. J Immunol Methods 47:25–30
O'Brien MC, Bolton WE (1995) Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry. Cytometry 19:243–255
Halldén G, Andersson U, Hed J et al (1989) A new membrane permeabilization method for the detection of intracellular antigens by flow cytometry. J Immunol Methods 124:103–109
Carson RT, Vignali DA (1999) Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay. J Immunol Methods 227:41–52
Picker LJ, Singh M, Zdraveski Z et al (1995) Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry. Blood 86: 1408–1419
Acknowledgment
This work was supported by funds from the Chinese National Natural Science Foundation No. 31271444 and No. 81201726 (Z. Shi), the Specialized Research Fund for the Doctoral Program of Higher Education No. 20124401120007 (Z. Shi), the Fundamental Research Fund for the Central University No. 21612407 (Z. Shi), NIH R15 No. 1R15CA143701 (Z.S. Chen), and St. John’s University Seed grant No. 579–1110 (Z.S. Chen).
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Qiu, JG., Mei, XL., Chen, ZS., Shi, Z. (2014). Cytokine Detection by Flow Cytometry. In: Vancurova, I. (eds) Cytokine Bioassays. Methods in Molecular Biology, vol 1172. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0928-5_21
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0928-5_21
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0927-8
Online ISBN: 978-1-4939-0928-5
eBook Packages: Springer Protocols