Application of image-based flow cytometry in cell phagocytosis

Phagocytosis is an important cellular process in innate immunity, development, and disease. Current methods for analyzing phagocytosis are largely limited to flow cytometry and manual image-based assays, providing limited information.

Here, we use image flow cytometry, an image-based and automated assay to rapidly quantitate binding and internalization stages during phagocytosis. PE-F4/80 labeled peritoneal macrophages were incubated with NBD-labeled PS beads, flow cytometry was first performed to analyze phagocytosis, and double positive cells were defined as phagocytic cells. It showed that the phagocytic percentage decreased from 63.83±6.30 % in ctrl to 32.66±4.79 % in Cytochalasin D treated (P<0.05), indicating that there were about 30% beads were bound to but not internalized into macrophage. The phagocytic level was also statisticaled by the Internalization wizard in image flow cytometry. The beads that overlapped with two peripheral pixels of phagocytes were considered as bound but not internalized; the rest was considered as internalized.

The results showed that the binding and internalization percentage were 64%, 36% respectively. Image flow cytometry offers the ability to automatically distinguish large amounts of image data into the binding and internalization within minutes, clearly demonstrating its potential value in investigating phagocytosis.

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