Quantifying Neutrophil Extracellular Traps

We examined NETs formation visually using a live-cell imaging system as reported previously ^{31 (link)}. Briefly, neutrophils on a 35-mm glass dish (Matsunami, Tokyo, Japan) were incubated with 500 nM Picogreen (Thermo Fisher Scientific, San Jose, CA, USA) for staining extracellularly-released DNA and 500 nM HySOx (Goryo chemical, Sapporo, Japan) for probing hypochlorous acid. Confocal fluorescence microscopy (FV-10; Olympus, Tokyo, Japan) was performed in a CO₂ incubator at 37 ˚C and the area of positive signal of Picogreen was quantified using ImageJ processing software (U. S. National Institutes of Health, Bethesda, MD, USA). NETs formation was also quantified by measuring the fluorescence intensity of Picogreen-labeled extracellular DNA from 1 × 10⁵ neutrophils in 300 μL culture medium. In some experiments, anti-MPO (Clone 2C7; BIO-RAD, Hercules, CA, USA) and anti-Histone H3 (Abcam, Cambridge, UK) antibodies were used to stain NETs components.