In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus

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Author: Morales, H
Author: Robert, J
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Methods Article

In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus

Heidi Morales¹ and Jacques Robert¹^*

¹ Department of Microbiology and Immunology, University of Rochester Medical Center. Rochester, NY 14642.

* To whom correspondence should be addressed: Jacques Robert, Department of Microbiology and Immunology, University of Rochester Medical Center. Rochester, NY 14642. USA. Phone: (585) 275 1722. Fax: (716) 473 9573. Email: jacques_robert@urmc.rrochester.edu

Biol. Proced. Online 2008;10:1-8. doi:10.1251/bpo137
Submitted: September 06, 2007; Accepted: September 26, 2007; Published: January 17, 2008.

Indexing terms: xenopus laevis; ranavirus; T-Lymphocytes.

Abbreviations: Ag, antigen; APBS, amphibian phosphate buffered saline; BrdU, bromodeoxyuridine; CFSE, carboxyfluorescein diacetate succinimidyl ester; FACS, fluorescence activated cell sorting; mAb, monoclonal antibody; MHC, major histocompatibility complex.

Figure 2 Enlarged

Fig. 2:

Proliferation of CD8+ T and IgM+ B cells 6 days post-FV3 infection detected by BrdU assay. Representative two-color flow cytometry analysis of splenocytes from uninfected control frogs (A), and frogs infected for 6 days (B). Splenocytes were surface stained for Xenopus MHC Class II (AM20), CD8 (AM22) or IgM (10A9) followed by APC-conjugated goat anti-mouse secondary antibody. Cells were then permeabilized, treated with DNase and stained with FITC-conjugated anti-BrdU mAb and analyzed by FACS. Twenty thousand total events were gated based on side and forward scatter profiles as in Fig. 1. The number in the upper right quadrant indicates the percent of double positive cells.

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