Optimization of phagocytosis assay in rainbow trout Oncorhynchus mykiss

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00027162%3A_____%2F18%3AN0000150" target="_blank" >RIV/00027162:_____/18:N0000150 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.was.org/Meetings/Default.aspx?code=Aqua18" target="_blank" >https://www.was.org/Meetings/Default.aspx?code=Aqua18</a>

DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Optimization of phagocytosis assay in rainbow trout Oncorhynchus mykiss

Popis výsledku v původním jazyce

AQUA 2018, Montpellier, Francie, 25.-29. 8. 2018 - lecture. Requirements for disease diagnosis increase along with the global aquaculture growth. An effective way of examining the fish immune system and its ability to actively react to stimulants is the usage of functional immunological assays. During phagocytosis assay, phagocytes are incubated with fluorescently labeled particles (e.g. zymosan, prepared from yeast cell walls). Peripheral blood is an optimal sample for this assay. Hemolysis in a hypotonic environment is a quick and cheap way of leukocyte isolation. The phagocyte representation and the intensity of phagocytic activity can then be measured by flow cytometry, which offers many advantages, in particular rapid examination of large numbers of cells and easy differentiation of individual cell populations. In fish, however, large variability needs to be taken into account. Phagocytic activity was also recorded in B-lymphocytes. The methodology varies between different authors – incubation time, incubation temperature and concentration of zymosan particles need to be optimized. Leukocytes were obtained from rainbow trout (Oncorhynchus mykiss) blood taken from the tail vein into a heparinized syringe. Incubation with zymosan particles (AF 488, Texas Red) was carried out in a CO2-free incubator. Leukocytes were isolated by hemolysis in a hypotonic environment (distilled water). Isolated cells were washed by centrifugation in PBS and fixed (Cell Wash, EDTA). As a second option, the cells were hemolysed first and then incubated with opsonized zymosan, medium (HBSS) and serum. Using a flow cytometer (BD LSRFortessa), the numbers of phagocyte cells and the intensity of phagocytic activity was evaluated. Propidium iodide was used to visualize dead cells. Incubation time, incubation temperature, appropriate zymosan particle concentration and volume of blood were optimized. Presently, best results were obtained with 1-hour incubation at 15°C. Incubation of 10 µl of blood and 1µl of zymosan AF 488 particles followed by hemolysis was proven to be most effective. More experiments are being performed to confirm these results.

Název v anglickém jazyce

Optimization of phagocytosis assay in rainbow trout Oncorhynchus mykiss

Popis výsledku anglicky

AQUA 2018, Montpellier, Francie, 25.-29. 8. 2018 - lecture. Requirements for disease diagnosis increase along with the global aquaculture growth. An effective way of examining the fish immune system and its ability to actively react to stimulants is the usage of functional immunological assays. During phagocytosis assay, phagocytes are incubated with fluorescently labeled particles (e.g. zymosan, prepared from yeast cell walls). Peripheral blood is an optimal sample for this assay. Hemolysis in a hypotonic environment is a quick and cheap way of leukocyte isolation. The phagocyte representation and the intensity of phagocytic activity can then be measured by flow cytometry, which offers many advantages, in particular rapid examination of large numbers of cells and easy differentiation of individual cell populations. In fish, however, large variability needs to be taken into account. Phagocytic activity was also recorded in B-lymphocytes. The methodology varies between different authors – incubation time, incubation temperature and concentration of zymosan particles need to be optimized. Leukocytes were obtained from rainbow trout (Oncorhynchus mykiss) blood taken from the tail vein into a heparinized syringe. Incubation with zymosan particles (AF 488, Texas Red) was carried out in a CO2-free incubator. Leukocytes were isolated by hemolysis in a hypotonic environment (distilled water). Isolated cells were washed by centrifugation in PBS and fixed (Cell Wash, EDTA). As a second option, the cells were hemolysed first and then incubated with opsonized zymosan, medium (HBSS) and serum. Using a flow cytometer (BD LSRFortessa), the numbers of phagocyte cells and the intensity of phagocytic activity was evaluated. Propidium iodide was used to visualize dead cells. Incubation time, incubation temperature, appropriate zymosan particle concentration and volume of blood were optimized. Presently, best results were obtained with 1-hour incubation at 15°C. Incubation of 10 µl of blood and 1µl of zymosan AF 488 particles followed by hemolysis was proven to be most effective. More experiments are being performed to confirm these results.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

40301 - Veterinary science

Projekt

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Návaznosti

V - Vyzkumna aktivita podporovana z jinych verejnych zdroju

Rok uplatnění

2018

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů