Investigation on hemocyte phagocytosis of Mytilus galloprovincialis through flow cytometry, micromethod fluorescence assay and confocal laser scanning microscopy

Flow cytometry, micromethod assay and confocal laser scanning microscopy have been applied in the study of the cellular defense system of mussel (Mytilus galloprovincialis), using yeast cells (zymosan) as phagocytosis stimuli and fluorescent probe, as 1,2,3-dihydrorhodamine (DHR), for measuring the synthesis of the reactive oxygen species (ROS). The flow citometry revealed the presence of two main hemocytes sub-populations in the mussel hemolymph, showing also the relation between hemocyte size, complexity and “respiratory burst” intensity. The micromethod assay demonstrated the oxidative activity of hemocytes during zymosan phagocytosis whereas the use of the SOD and L-NMMA increased the fluorescence, confirming the main role of the hydrogen peroxide in the DHR oxidation. In addition, the confocal microscopy permitted to valuate the ROS synthesis inside the cellular organelles involved in the phagocytosis. In conclusion, these original techniques could represent useful tools for valuating the cellular immune system of marine invertebrates, both in research approach and in applied studies.[...