Springer Protocols: Abstract: Dual-Color ELISPOT Assay for Analyzing Cytokine Balance

Dual-Color ELISPOT Assay for Analyzing Cytokine Balance

By: Yoshihiro Okamoto², Mikio Nishida²

Abstract

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A dual-color enzyme-linked immunospot (ELISPOT) assay enabled us to analyze three kinds of cytokine-secreting cells simultaneously. T helper (Th) cells can be subdivided into at least two distinct functional subsets based on their cytokine secretion profiles. The first type of clones (Th1) produces interleukin (IL)-2 and interferon (IFN)-γ but not IL-4 or IL-5. The second type of clones (Th2) produces IL-4 and IL-5 but not IL-2 or IFN-γ. Furthermore, the presence of the third type (Th0) cell, which is a precursor of Th1 or Th2 cells, has been demonstrated to produce both Th1- and Th2-type cytokines. The dual-color ELISPOT assay is developed to differentiate these three subtypes of Th cells in an identical well. In the system, the red spots corresponding to IL-2 secreting cells (Th1) were developed with horseradish peroxidase and amino-ethyl-carbazole/H₂ O₂. The light blue spots corresponding to IL-4-secreting cells (Th2) were developed with alkaline phosphatase and Vector blue (chromogenic substrate for alkaline phosphatase). The mixed colored (indigo) spots corresponding to both kinds of cytokine-secreting cells (Th0 cells) were developed with both chromogenic substrates. With this system, we could detect the IL-2- and/or IL-4-secreting cells simultaneously in a murine spleen cell or human peripheral mononuclear cell preparation.

Affiliation(s): (2) Division of Health Care Pharmacy, Faculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya, Aichi, Japan

Book Title: Handbook of ELISPOT: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 302 | Pub. Date: Mar-15-2005 | Page Range: 263-272 | DOI: 10.1385/1-59259-903-6:263

Subject: Immunology

Key Words: Dual-color enzyme-linked immunospot assay - interleukin-2 - interleukin-4 - cytokine balance - mouse - human

References

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