Sytox green reagent

To quantify NETs in the cell culture supernatant and plasma, we used the PicoGreen dsDNA Quantification Kit (Invitrogen, Carlsbad, CA, USA) and a capture ELISA based on myeloperoxidase (MPO) associated with DNA [18 (link)]. For ELISA analysis of NET concentration in plasma, 1 μg/mL anti-MPO mAb was used as a capture antibody with Cell Death Detection ELISA (Roche, Indianapolis, IN, USA) according to the instructions.
NET formation was also quantified by confocal microscopy. PMNs were allowed to settle on glass coverslips precoated with poly-l-lysine (#354085, Corning, NY, USA) for 30 min prior to being treated for a specific period of time. PMNs were incubated with 1 μM SYTOX Green reagent (#S7020, Invitrogen, USA) at 37 °C for 10 min. Nuclei were counterstained using DAPI, and the cells were mounted in Antifade Mounting Medium (#P0126, Beyotime Biotechnology, Shanghai, China) for imaging with a confocal microscope. For each slice, 5 random fields were captured and analysed. NET-positive cells and NET area were quantified using ImageJ software v.1.3.7. Only structures depicting NET morphology and positive for SYTOX Green were selected for area quantification, and intact granulocyte nuclei were excluded from the analysis.

Aliquots of 10⁶ neutrophils/mL in HBSS/Ca⁺² (1 mL) was prepared in microtubes following the planned groups. The sytox green reagent (Invitrogen) was added to each tube at a final concentration of 0.2 µM. Cells exposed to HBSS 1× or Triton 100× were applied as negative and positive controls, respectively. U87MG conditioned media was produced according to the following parameters: after reaching 85% confluency in T75-flask, 10 mL of DMEM with 0.5% or 10% FBS were kept for 72 h, at 37°C, 5% CO₂. PMA (100 nM) and LPS (1 μg/mL) groups were performed as control. PMNs were incubated for 3 h and 200 µL of each condition was transferred to a separate well of a 96-well black plate. Fluorescence was quantified at excitation/emission wavelengths of 488/520 nm with a microplate reader (26 (link)). The results were expressed as relative fold change by dividing the average of the experimental condition by the average of the untreated condition (negative control).

The dormant cells of wild type and ∆pspA were identified by time-lapse microscopy using the Flow Cell System (Bioptechs FCS2, USA). Cells from the middle stationary phase were harvested and tipped on a gel pad containing 2% (wt/vol) low melting temperature agarose in LB medium. Then, cell growth was recorded under the bright-field microscope (Zeiss Axio Observer Z1, Germany) for 5 h at 37°C. Dormant cells were defined as cells non-proliferating for 5 h during observation.
For the SYTOX green staining to distinguish with the viable cells and dead cells, the ∆pspA cells from the middle stationary phase were washed with phosphate-buffered saline (PBS) and diluted to an OD₆₀₀ of 0.2 in PBS with 5 µM SYTOX green reagent (Invitrogen, S7020). Then, the cells were incubated at 37°C for 15 min in the dark. After staining, the cells were washed with PBS to remove the SYTOX green reagent, resuspended in LB and collected for imaging.