Optimisation of the lymphocyte proliferation assay in rainbow trout (Oncorhynchus mykiss)

H Minarova, M Palikova, J Mares, E Syrova, J Blahova, M Faldyna and P Ondrackova
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H Minarova: Department of Ecology and Diseases of Game, Fish and Bees, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic
M Palikova: Department of Ecology and Diseases of Game, Fish and Bees, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic
J Mares: Department of Zoology, Fisheries, Hydrobiology and Apiculture, Faculty of AgriSciences, Mendel University in Brno, Brno, Czech Republic
E Syrova: Department of Ecology and Diseases of Game, Fish and Bees, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic
J Blahova: Department of Animal Protection, Welfare and Behaviour, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic
M Faldyna: Department of Immunology, Veterinary Research Institute, Brno, Czech Republic
P Ondrackova: Department of Immunology, Veterinary Research Institute, Brno, Czech Republic

Veterinární medicína, 2019, vol. 64, issue 12, 547-557

Abstract: The lymphocyte proliferation assay is a valuable method used for the evaluation of the fish immune system. However, there are many variations and optimal results are not always obtained. Unification is necessary to ensure the comparability between different studies. The aim of this study was to optimise the lymphocyte proliferation assay in rainbow trout (Oncorhynchus mykiss). This goal included the determination of the optimal incubation length, serum type, incubation temperature, type of mitogen and its concentration, and anticoagulant. The peripheral blood and head kidney lymphocytes were isolated by density gradient centrifugation. Subsequently, the cells were incubated for 3-8 days with different mitogens (pokeweed mitogen 5, 10 and 50 µg/ml, concanavalin A 1, 10 and 20 µg/ml, phytohaemagglutinin 25, 50 and 100 µg/ml, lipopolysaccharide 1, 50 and 100 µg/ml). The use of the different serum types (foetal bovine serum, trout serum), incubation temperatures (10-20 °C) and anticoagulants (heparin, EDTA) was compared. Labelled thymidine was used to evaluate the assay. The best results were obtained after seven days of incubation at 15 °C with foetal bovine serum (FBS). The head kidney lymphocytes showed the highest proliferative response with 50 µg/ml phytohaemagglutinin. With the peripheral blood lymphocytes (heparin and EDTA), the best results were obtained with 50 µg/ml pokeweed mitogen. The highest proliferation levels were detected with heparinised blood. In conclusion, optimisation of this assay contributes to the improved assessment of the rainbow trout immune function.

Keywords: fish; mitogen; stimulation; thymidine; transformation (search for similar items in EconPapers)
Date: 2019
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Persistent link: https://EconPapers.repec.org/RePEc:caa:jnlvet:v:64:y:2019:i:12:id:98-2019-vetmed

DOI: 10.17221/98/2019-VETMED

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