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The aim of this study is to modify the flow cytometric methods for avian heterophil that used to analyzeneutrophil functions. Within the aim of the project, we tested the amount of blood for acquiring theheterophil and the storage duration of the blood before the analysis and the time of centrifugation. Also,we tested the amounts of cell suspension and dihydrordamine-123 (DHR-123) during the flow cytometricanalysis. We reviewed the amount of porbol miristat asetat (PMA) used to stimulate the oxidative burstand the amount of formyl methionyl-leucyl-phenylalanine (fMLP) used to stimulate chemotaxic activity.Experiments on the incubation temperature and incubation duration were also performed. The resultsshowed that 0.5-3 ml of blood could be used to detect heterophil functions and it would be ideal to study infresh blood samples. However, it also showed that the stored blood can be used for a maximum of 8 hoursat +4 degrees. In order to isolate the cells, centrifugation of blood samples for 30 minutes would besufficient, and it would be appropriate to use 30μL from the cell suspension. DHR-123, which is used as achemical probe to measure heterophil functions, had to be used in 2μL, and when used excessively, itaffected the heterophil functions negatively. In addition, it was seen that using 2μL each of fMLP, which isused as an oxidative burst stimulant, and PMA as a stimulant of chemotaxic activity was sufficient. It wasconcluded that the incubation at 41 ° C for 5 minutes after stimulating the heterophil would also besufficient. As a result, it was thought that this study could be used to isolate heterophil and to analyze withflow cytometry and to contribute further research and clinical studies in poultry. |
