Quantitative Analysis of Latex Beads Phagocytosis by Human Macrophages Using Imaging Flow Cytometry with Extended Depth of Field (EDF)

The existing methods of quantitative analysis of phagocytosis are characterized by a number of limitations. The usual method of manually counting phagocytosed objects on photographs obtained by confocal microscopy is very labor-intensive and time-consuming. As well, the resolution of conventional flow cytometry does not allow the fluorescence detection of a large number of phagocytosis objects. Thus, there is a need to combine the rapid analysis by flow cytometry and the visualization capability by confocal microscopy. This is possible due to imaging flow cytometry. However, until now, no protocols have allowed one to quantify phagocytosis at its high intensity. The present paper presents the developed and tested algorithm for assessing the level of phagocytic activity using flow cytometry with visualization and IDEAS software.