A phagocytosis assay is one of the most commonly used functional immunological methods. There are many possible ways of assessing leucocytes and their ability to ingest different particles. The aim of this study was to optimise the phagocytosis assay in rainbow trout (Oncorhynchus mykiss) using labelled zymosan particles (Alexa Fluor 488 and Texas Red conjugate). Whole blood was incubated with the particles under different conditions and leucocytes were subsequently isolated by haemolysis in a hypotonic environment. The effect of the different incubation time, temperature, blood volume and dilution on the phagocytic activity was evaluated by flow cytometry. Our experiments showed that the incubation for at least 2 h at 15 °C provided optimal results, while the blood volume and dilution had no significant effect. The optimised assay will be used for the examination of fish health and in further experimental studies.

Department of Animal Protection and Welfare and Veterinary Public Health Faculty of Veterinary Hygiene and Ecology University of Veterinary Sciences Brno Brno Czech Republic

Department of Ecology and Diseases of Zoo Animals Game Fish and Bees Faculty of Veterinary Hygiene and Ecology University of Veterinary Sciences Brno Brno Czech Republic

Department of Infectious Diseases and Preventive Medicine Veterinary Research Institute Brno Czech Republic

Department of Zoology Fisheries Hydrobiology and Apiculture Faculty of AgriSciences Mendel University in Brno Brno Czech Republic

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