In vitro cocultures of macrophages and apoptotic cells (ACs) provide a practical and useful tool to study efferocytosis. Here, we describe a method for automated quantification and imaging of recognition and engulfment of apoptotic cells by primary macrophages using imaging flow cytometry (IFC). IFC-based analysis allows us to successfully quantify efferocytosis, clearly distinguishing phagocytic from nonphagocytic macrophages and, more importantly, from those in recognition stage, which is not achievable by standard flow cytometrical analysis. To this end, we established a universally employable analysis pipeline to address efferocytosis that can be easily adapted to any macrophage population from samples of different origins.

Our knowledge of the intestinal immune system of fish is rather limited compared to mammals. Very little is known about the immune cells including the phagocytic cells in fish intestine. Hence, employing imaging flow cytometry and RNA sequencing, we studied adherent cells isolated from healthy Atlantic salmon. Phagocytic activity and selected gene expression of adherent cells from the distal intestine (adherent intestinal cells, or AIC) were compared with those from head kidney (adherent kidney cells, or AKC). Phagocytic activity of the two cell types was assessed based on the uptake of Escherichia coli BioParticlesTM. AIC showed phagocytic ability but the phagocytes were of different morphology compared to AKC. Transcriptomic analysis revealed that AIC expressed genes associated with macrophages, T cells, and endothelial cells. Heatmap analysis of selected genes indicated that the adherent cells from the two organs had apparently higher expression of macrophage-related genes. We believe that the adherent intestinal cells have phagocytic characteristics and high expression of genes commonly associated with macrophages. We envisage the possibilities for future studies on enriched populations of adherent intestinal cells.

Imaging flow cytometry (IFC) is a powerful tool which combines flow cytometry with digital microscopy to generate quantitative high-throughput imaging data. Despite various advantages of IFC over standard flow cytometry, widespread adoption of this technology for studies in aquatic sciences is limited, probably due to the relatively high equipment cost, complexity of image analysis-based data interpretation and lack of core facilities with trained personnel. Here, we describe the application of IFC to examine phagocytosis of particles including microplastics by cells from aquatic animals. For this purpose, we studied (1) live/dead cell assays and identification of cell types, (2) phagocytosis of degradable and non-degradable particles by Atlantic salmon head kidney cells and (3) the effect of incubation temperature on phagocytosis of degradable particles in three aquatic animals-Atlantic salmon, Nile tilapia, and blue mussel. The usefulness of the developed method was assessed by evaluating the effect of incubation temperature on phagocytosis. Our studies demonstrate that IFC provides significant benefits over standard flow cytometry in phagocytosis measurement by allowing integration of morphometric parameters, especially while identifying cell populations and distinguishing between different types of fluorescent particles and detecting their localization.