Hysox

The concentration of H₂O₂ in pure water and culture medium generated by EES treatment was measured using the Amplex Red probe (Invitrogen, Carlsbad, CA, USA). EES treatment was performed on 1.5 ml pure water or culture medium without cells, and 50 μl of the treated solutions was transferred to individual wells of a 96-well transparent plate. To generate a standard curve, H₂O₂ solution of 0 to 10 μM was prepared. After adding the Amplex Red reagent to each well, the microplate was incubated at room temperature for 30 min. Then, fluorescence was measured using a multiwell plate reader (TECAN Infinite 200 PRO, Männedorf, Switzerland) with excitation/emission filters of 550/585 nm.
Detection of other intracellularly induced ROS was also performed using fluorescent probes. Cells were cultured for 3 days and then stained with OxiOrange (excitation: 543 nm, emission: 577 nm; Goryo Chemical, Sapporo, Japan) or HySOx (excitation: 555 nm, emission: 575 nm; Goryo Chemical), which react with intracellular HO· and hypochlorous acid (HClO), respectively, and emit strong fluorescence. After staining for 30 min at 37°C, cells were washed with PBS, and EES treatment was conducted with 0.5 ml culture medium, and then intracellularly induced ROS were observed by fluorescence microscopy (Axio Observer, Carl Zeiss, Jena, Germany). Nuclei were stained with DAPI (4′6-diamidino-2-phenylindole).

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.