Quantification of Neutrophil Extracellular Trap (NET)Formation in Healthy Subjects

Polymorphonuclear neutrophils (PMNs) play a crucial role in the first line of host defense against foreign antigen or invading pathogen. It was discovered as a novel microbial killing mechanism of neutrophils, called neutrophil extracellular trap (NET) formation. NETs are identified as web-like extracellular structures composed of decondensed nuclear DNA, histones, and antimicrobial granules. Upon neutrophil activation, NETs are released as an antimicrobial activity that can entrap and kill microbes extracellularly. Nevertheless, NETs may function as double-edged swords, serving as an adverse pathological outcome in excessive NET formation such as in severe sepsis or auto-inflammatory diseases. The aim of this study was investigation of a potential technique for quantification of NETs (or extracellular DNA backbone) released by human neutrophils in culture supernatants using Hoechst 33258 staining. The results revealed that, in the range of 340 to 370 nm, Hoechst 33258 had maximum fluorescence intensity at 365 nm excitation wavelength in dsDNA solutions detected by spectrofluorometer. Additionally, Hoechst 33258 at 1 µ g/mL was an optimal concentration to quantify the amount of NET releases in vitro . The level of NETs was significantly elevated in phorbolmyristate acetate (PMA)-stimulated neutrophils as compared to that in medium control, suggesting that NETs could be efficiently quantitated with Hoechst 33258. This work strengthens our understanding that Hoechst 33258 should prove useful as a simple method to apply in NET-DNA quantification in vivo in further studies.