NET Quantification and ROS detection
NET quantification was performed as previously described (18). In brief, the cells were seeded at 5.2 × 10^4 cells per well in a 96-well plate in the culture media and were preincubated with antibiotics for 2 h at 37 °C in 5% CO2. Then, PMA (100 nM) was added in the presence of 5 μM SYTOX Green cell-impermeable nucleic acid stain (Life Technologies). The fluorescence intensity (RFU) was measured using a SpectraMax i3x fluorescence microplate reader (Molecular Devices LLC) at specific time intervals for up to 200 min after the activation of the cells. For the inhibitory assays, an extra 1-h preincubation with DPI or Pyro was needed. The NETotic index was used to calculate the index of NET formation and is expressed as the RFU ratio of each group. Some cells were lysed with 0.5% Triton X-100 at each time point, and this sample represented 100% DNA release.
In order to quantify reactive oxygen species (ROS) generation, isolated PMNs were suspended in RPMI 1640 medium containing 1% HEPES and pretreated with Dihydrorhodamine 123 (DHR123, 1 ummol/L, Beyotime, Shanghai, China) for 30 minutes. Then washing PMNs three times with PBS after centrifugation. The same number of PMNs were then resuspended with PBS and transferred into a black 96-well microplate. Fluorescence intensity was quantified every 10 minutes after treated with PMA, which using SpectraMax i3x fluorescence microplate reader (Molecular Devices LLC) at an excitation wavelength of 488nm and an emission wavelength of 530nm.