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

Abstract

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  µg/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.