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Stepping up ELISpot: Multi-Level Analysis in FluoroSpot Assays
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Cells 2014, 3(4), 1116-1130; doi:10.3390/cells3041116

Triple Cytokine FluoroSpot Analysis of Human Antigen-Specific IFN-γ, IL-17A and IL-22 Responses

1
Mabtech AB, Box 1233, SE-131 28 Nacka Strand, Sweden
2
Department of Immunology, Stockholm University, SE-106 91 Stockholm, Sweden
*
Author to whom correspondence should be addressed.
Received: 15 October 2014 / Revised: 10 November 2014 / Accepted: 21 November 2014 / Published: 27 November 2014
(This article belongs to the Special Issue ELISPOT Research)
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Abstract

The involvement of T-helper (Th)1, Th17 and Th22 cell subsets, in immunity, as well as in pathological inflammatory reactions, makes it important to determine their relative proportion. A triple FluoroSpot detecting the hallmark cytokines of Th1 (IFN-γ), Th17 (IL-17A) and Th22 (IL-22) was developed and evaluated using human peripheral blood mononuclear cells from healthy donors incubated with tetanus toxoid, Candida albicans extract, mycobacterial purified protein derivative or medium only. Antigen stimulation yielded mainly cells secreting IFN-γ, IL-17A or IL-22 alone but lower proportions of double-secreting cells were also found; triple-secreting cells were rare. The response to C. albicans contrasted in that higher proportions of IL-17A single secreting as well as co-secreting cells, in particular IL-17A/IL-22, were found. The FluoroSpot analysis correlated well with single cytokine ELISpot assays ran in parallel and the methods displayed a comparable sensitivity. The results demonstrate the functionality of the FluoroSpot assay for simultaneous analysis of distinct Th1, Th17, Th22 as well as intermediate cell populations. The method provides a mean for a simple and rapid analysis of the involvement of these cells in immunity and disease. View Full-Text
Keywords: FluoroSpot; ELISpot; T-helper cell subsets; IFN-γ; IL-17A; IL-22; Th1; Th17; Th22 FluoroSpot; ELISpot; T-helper cell subsets; IFN-γ; IL-17A; IL-22; Th1; Th17; Th22
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Dillenbeck, T.; Gelius, E.; Fohlstedt, J.; Ahlborg, N. Triple Cytokine FluoroSpot Analysis of Human Antigen-Specific IFN-γ, IL-17A and IL-22 Responses. Cells 2014, 3, 1116-1130.

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