Discussion

Phagocytosis - engulfment of solid foreign particles by cells - is commonly detected using flow cytometry or fluorescence microscopy. Target bioparticles labelled with fluorophores are engulfed upon binding to the surface of phagocytic cells; at a given end-point non-engulfed bioparticles are removed or quenched and the cells fixed and analysed to determine the proportion containing the fluorescent label. Here we describe a simple mix-and-read assay that uses IncuCyte ZOOM® technology, coupled with bioparticles conjugated to pH sensitive probes, to quantify real time phagocytic uptake in live cells.
Murine macrophage J774A.1 cells were seeded into 96-well microtitre plates. The following day sterile pHrodo™ conjugated bioparticles derived from bacteria or yeast (E. coli, S. aureus, S. cerevisiae) were added to the J774A.1 cells maintained in standard growth media. The assay plates were placed in an IncuCyte ZOOM® imaging system and high definition phase contrast and fluorescent images were captured at 15 or 30 minute intervals for up to 48 h (20x or 10x magnification). As the bioparticles were engulfed and reached the lower pH environment of the phagosomes increasing fluorescent intensity was observed, initially as punctate staining before becoming more diffuse throughout the cytosol at later time-points (>4 h). Quantification of the time-course of fluorescent intensity demonstrated the rate and magnitude of phagocytosis was both cell number- and bioparticle number-dependent. Importantly, the cell number could be reduced to as low as 1000 cells per well whilst maintaining a sufficient signal window. Pharmacological disruption of phagocytosis was quantified; the actin polymerase inhibitor cytochalasin D yielding a concentration-dependent reduction of pHrodo conjugated E. coli bioparticle uptake; IC50 values of 1.17 µM or 0.53 µM for green or red fluorophores, respectively.
Key benefits of this approach include (i) the ability to visualize and monitor phagocytosis in real time over many hours or days (ii) low cell and bioparticle usage and (iii) highly sensitive image-based detection of phagocytosis from fluorescence background subtracted images. In summary we provide a phagocytosis assay protocol in a microtitre plate format that is suitable for screening and enables rapid compound profiling.