Morales H and Robert J (2008), In vivo and in vitro techniques for comparative...

XB-ART-37485

Biol Proced Online 2008 Jan 17;10:1-8. doi: 10.1251/bpo137.

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

Morales H , Robert J .

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Activation of lymphocytes in mammals is often quantified by measuring the amount of proliferation during the expansion phase of an immune response. Bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assays are some of the techniques widely used in mammalian studies of pathogen-induced proliferation and provide a convenient way of quantifying the cellular response. We have extended the use of these proliferation assays to the amphibian Xenopus laevis. We have developed this species as a valuable comparative model to study immunity against a well-known amphibian pathogen, Frog Virus 3 (FV3). Fluorescence activated cell sorting was used to assess the level of BrdU incorporation of lymphocytes in vivo and CFSE dilution in an in vitro activation assay. Both techniques have shown that splenic lymphocytes proliferate specifically upon FV3 challenge. This indicates that common methods for detection of proliferation upon immunologic challenge are easily applied to other vertebrate species, as it highlights the evolutionary conservation of the proliferative nature of immune responses throughout vertebrate phyla.

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Species referenced: Xenopus laevis
Genes referenced: cd8a ighx mhc1a

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	Fig. 1 Detection of Xenopus splenocyte proliferation 6 days post-FV3 infection by BrdU assay. Uninfected control frogs and frogs infected for 6 days with FV3 were treated with BrdU 48 hrs before the assay. Cells were then permeabilized, treated with DNase, stained with FITC-conjugated anti-BrdU mAb, and analyzed by FACS (Representative of six different experiments). Ten thousand total events were gated based on side and forward scatter profiles (A, C). The arrows depict cells with higher forward scatter that increase in number in the infected sample. The percent of gated BrdU positive cells is shown in the histograms (B, D).
	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.
	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.

References [+] :

Carayon, Identification of DNA-replicating lymphocyte subsets using a new method to label the bromo-deoxyuridine incorporated into the DNA. 1992, Pubmed