Phagocytosis is an essential process of the immune system to eliminate cellular debris and pathogens. It is a specific form of endocytosis involving vesicular internalization. Phagocytic cells such as macrophages can be attracted to pathogens or cellular debris and engulf the material to be trapped in an internal vesicle called phagosome. The phagosome would fuse with the lysosome to form phagolysosome, where enzymes and toxic peroxides can digest the pathogen or cellular debris at a low pH value. The host-defense activation of phagocytosis is by attachment of mammalian immune cells to Pathogen-associated molecular patterns (PAMPS), which can lead to NF-κB activation.
Traditionally, live cell analysis of phagocytosis can be qualitatively examined via standard optical microscopy to observe the engulfment of bacteria or cellular debris. In addition, phagocytosis can also be quantitatively measured by incubating phagocytes with particles bound with serum or IgG. After incubation, the excess particles are washed away, leaving only phagocytes with engulfed particles. These cells are lysed and release the engulfed material, which can then be examined by using microplate reader or western blot analysis to quantify the level of phagocytosis. However, these methods are time-consuming and require experienced researchers to obtain meaningful results.
By using the Celigo imaging cytometer, phagocytosis can be measured using a pH sensitive label called pHrodo. The labels will only fluoresce when they are trapped in the phagolysosome where the pH values are low. Therefore, by measuring the amount of fluorescence or percentage of cells fluorescing, one can use the Celigo to determine the fluorescent intensities of the target cell populations and measure the level of phagocytosis.