In the above figure, a neutrophil is seen phagocytosing a FITC-labelled organism and then external organisms are quenched using trypan blue.
One useful method described uses fluorescein heat-killed Candida albicans, and after phagocytosis is complete, ethidium bromide (EB) (50 ug/ml) is added. Analysis using ultraviolet (UV) excitation with red and green emission reveals green internalized organisms, while surface attached, but non-internalized organisms are red. This procedure utilizes the phenomenon of resonance energy transfer between FITC and ethidium bromide. EB does not penetrate the cell membrane so only the external organisms are affected by the providing discrimination between internal and external organisms. This assay can be performed on clinical analyzers with simple laser configurations.
Flow cytometry can also be used for evaluating neutrophil function in the phagocytosis of fluorescent-labeled viruses. FITC-labeled Herpes simplex viruses (HSV) have been shown to be phagocytosed by human neutrophils and both internalization and surface binding were determined by flow cytometry. Surface bound virus fluorescence was quenched using a trypan blue quenching procedure.
Pinocytosis can also be a useful measure of cell function and several well-defined assays have been developed for flow cytometry. fMLP stimulated pinocytosis studies have demonstrated a linkage between the initial phase of pinocytosis and the characteristic shape changes observed in activated neutrophils. This assay uses FITC-dextran and is relatively simple to establish. A number of concomitant effects such as pH changes, kinetics of the responses, temperature and ionic concentration effects can be evaluated in this manner.
