DNA Area and NETosis Analysis (DANA): a High-Throughput Method to Quantify Neutrophil Extracellular Traps in Fluorescent Microscope Images

Background: Neutrophil extracellular traps (NETs), extracellular structures composed of decondensed chromatin and antimicrobial molecules, are released in a process called NETosis. NETs, which are part of normal host defense, have also been implicated in multiple human diseases. Unfortunately, methods for quantifying NETs have limitations which constrain the study of NETs in disease. Establishing optimal methods for NET quantification holds the potential to further elucidate the role of NETs in normal and pathologic processes. Results: To better quantify NETs and NET-like structures, we created DNA Area and NETosis Analysis (DANA), a novel ImageJ/Java based program which provides a simple, semi-automated approach to quantify NET-like structures and DNA area. DANA can analyze many fluorescent microscope images at once and provides data on a per cell, per image, and per sample basis. Using fluorescent microscope images of Sytox-stained human neutrophils, DANA quantified a similar frequency of NET-like structures to the frequency determined by two different individuals counting by eye, and in a fraction of the time. As expected, DANA also detected increased DNA area and frequency of NET-like structures in neutrophils from subjects with rheumatoid arthritis as compared to control subjects. Using images of DAPI-stained murine neutrophils, DANA (installed by an individual with no programming background) gave similar frequencies of NET-like structures as the frequency of NETs determined by two individuals counting by eye. Further, DANA quantified more NETs in stimulated murine neutrophils compared to unstimulated, as expected. Conclusions: DANA provides a means to quantify DNA decondensation and the frequency of NET-like structures using a variety of different fluorescent markers in a rapid, reliable, simple, high-throughput, and cost-effective manner making it optimal to assess NETosis in a variety of conditions.