Optimizing Studies of Phagocytic Activity by Flowsight Cytometry in Amphibians

Abstract. Phagocytosis is a primary and highly conserved mechanism for clearing the extracellular milieu from pathogens and debris. In amphibians, the lack of antibodies for characterizing the different phenotypes of phagocytic cells has impaired the study of the phagocytic process. We used conventional and flowsight cytometry to determine immune cells' phagocytic activity from the blood and peritoneum of toads by in vitro and in vivo assays. Macrophage-like and neutrophil-like cells were clustered and analyzed according to cell morphology and the number of internalized zymosan particles by flowsight cytometry. We identified peritoneal and blood phagocytes (macrophage-like/monocyte-like and neutrophil-like) and lymphocyte-like cells. Besides, we observed monocyte-like/macrophage-like and neutrophil-like cells engulfing up to seven zymosan particles. Assessing the phagocytic activity from blood and peritoneal phagocytes using in vitro and in vivo assays brings better insights into phagocytosis in amphibian immune cells from distinct body compartments and approaches. Moreover, it is worth highlighting the importance of morphologically identifying the cells and evaluating the number of internalized particles by flowsight cytometry, a valuable asset to further explore phagocytosis and other cellular processes in amphibians under field and laboratory conditions.