Our research focuses on neutrophils, which are essential first responders in the immune system and play a significant role in various diseases. Over the past two decades, it has become clear that neutrophils contribute to the development of cancer, autoimmune diseases, and other inflammatory conditions by disrupting immunoregulation. This includes forming neutrophilic extracellular traps, nets, web-like structures that respond to inflammation and could be targeted for therapy in these diseases.
However, despite some promising molecules targeting nets, in development there is still no approved therapy that specifically affects this mechanism. This is at least partially attributable to the lack of an objective, unbiased, reproducible, and high-throughput quantification method for net formation. Our protocol employs dual-color live cell imaging to analyze neutrophil behavior using membrane permeable and impermeable dyes for precise net formation tracking.
This method distinguishes between net-forming and healthy neutrophils based on membrane integrity and provides clear differentiation of cell death types through morphological changes absorbed in phase contrast imaging. This method overcomes the problems of previously-reported techniques to quantify net formation and provides an efficient, reproducible, and accurate net quantification in an automated manner. This method will help in neutrophil-targeted drug development by giving the ability to screen multiple therapeutic targets against net formation in a high-throughput manner.
