High-throughput assays show timescale of phagocytic success depends on target toughness

A biofilm is a community of bacteria bound together in a matrix of extracellular polymeric substances (EPS) that can be produced by constituent bacteria or incorporated from the environment. The biofilm matrix protects constituent bacteria from external threats such as antibiotics and the immune system. Neutrophils are phagocytic white blood cells which clear infections by engulfing free-swimming bacteria, but are too small to engulf whole biofilms. Here we validate flow cytometry as high-throughput technique to measure phagocytic engulfment from populations of 20,000 neutrophils per experiment. Rapid data collection allows us to extend our study to neutrophils isolated from the whole blood of three healthy adult volunteers. We investigate phagocytic success using mechanically tunable polyacrylamide hydrogels infused with fluorescent microbeads as a model for bacterial biofilms. The tunability of polyacrylamide gels allows us to investigate the effects of toughness and yield strain independently from elastic modulus. We find that extended incubation times of up to 6 hours allow neutrophils to overcome low toughness of hydrogels and successfully phagocytose the imbedded beads, regardless of the gels’ elastic modulus. We begin extending these techniques to measure phagocytic success of neutrophils after incubation with live biofilms utilizing antibiotics to kill non-internalized bacteria, and pH-dependent stains which fluoresce brightly in acidic environments, e.g. inside the phagolysosome of a neutrophil.