Methods for Quantifying Neutrophil Extracellular Traps on Biomaterials.

Neutrophils rapidly accumulate at sites of inflammation, including biomaterial implantation sites, where they can modulate the microenvironment toward repair through a variety of functions, including superoxide generation, granule release, and extrusion of neutrophil extracellular traps (NETs). NETs are becoming increasing implicated as a central player in the host response to a biomaterial, and as such, there is a need for reliable in vitro methods to evaluate the relative degree of NETs and quantify NETs on the surface of biomaterials. Such methods should be relatively high throughput and minimize sampling bias. In this chapter, we describe two procedures, (1) fluorescent image analysis and (2) a NETs-based ELISA, both of which have been specifically optimized to quantify NETs generated from human neutrophils on electrospun polydioxanone templates. Both methods are valid and also compatible with tissue culture plastic, but have a variety of advantages and disadvantages. Therefore, both methods can be used to concomitantly study NETs on the surface of a biomaterial. Finally, while these methods were developed for electrospun templates in a 96-well cell culture plate, they may be easily adapted to a large scale and for other biomaterials, including but not limited to metallics, ceramics, and natural and synthetic polymers.