Abstract
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.
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© 2005 Humana Press Inc., Totowa, NJ
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Okamoto, Y., Nishida, M. (2005). Dual-Color ELISPOT Assay for Analyzing Cytokine Balance. In: Kalyuzhny, A.E. (eds) Handbook of ELISPOT. Methods in Molecular Biology™, vol 302. Humana Press. https://doi.org/10.1385/1-59259-903-6:263
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DOI: https://doi.org/10.1385/1-59259-903-6:263
Publisher Name: Humana Press
Print ISBN: 978-1-58829-469-2
Online ISBN: 978-1-59259-903-5
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