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 3 Enlarged

Fig. 3:

Detection of Xenopus primed splenocyte proliferation induced in vitro by FV3 infected FV3 peritoneal leukocytes using the CFSE assay. Splenocytes from an uninfected control frog or a frog primed 3 weeks before the assay by infection with FV3 were CFSE stained and co-cultured for 72 hours with FV3 infected peritoneal leukocytes obtained from the same frogs 3 days before the assay. Total culture was surface stained for MHC class II (AM20) or CD8 (AM22) and analyzed by FACS. A time zero analysis shows initial CFSE staining of splenocytes prior to co-culture. Analysis at 72 hrs (B) was done on gated population in the side scatter dot plot (A). Numbers in upper left quadrant indicates the percent of CD8 or Class II positive cells with diluted CFSE.

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