Sytox green dye

HeLa and BMDMs were seeded in a 12‐well plate and grown to 60% confluence before transfection. Transfection of C. albicans purified Sce1 protein was performed by PULSin protein delivery reagent (Polyplus‐transfection) with a total concentration of 2 µg/ml Sce1. The effectiveness of the method was confirmed by transfection of R‐phycoerythrin, a red fluorescent protein used as a positive control (Figure S8C). Exogenous expression of Sce1 in HeLa cells was assessed by transfection of pCDH plasmids expressing Flag‐tagged full‐length or N/C terminus of Sce1 into HeLa cells. To evaluate Sce1‐induced mammalian cell death, SYTOX Green dye or PI (Thermo Fisher Scientific) was added after the indicated time points. After incubation in the dark for 10 min, micrograph images were collected using an Olympus fluorescence microscope (Olympus IX73) equipped with 20× and 40× objective. Results were presented as percentages of SYTOX Green‐ or PI‐positive cells versus total live cells. To evaluate cell apoptosis, an Annexin V‐FITC/PI (Transgene) apoptosis detection kit was used. The Annexin V‐FITC/PI stained apoptotic cells were quantified and grouped by flow cytometry (BD Fortessa).

WT, Stat4^–/–, and Stat4^fl/flLysM^cre mice were injected with 3% of thioglycolate broth (Thermo Fisher Scientific) or PBS as a control i.p. to elicit a robust influx of neutrophils into the PC. After 6–72 hours, the peritoneal lavage was collected to investigate the morphological changes and immune responses of neutrophils. A cell-impermeable, DNA-binding Sytox Green dye (Thermo Fisher Scientific, S7020) was used for measuring the NET release. After filtering out the debris with a mesh, neutrophils were analyzed using Attune flow cytometer (Applied Biosystems, Thermo Fisher Scientific). Because Sytox Green expresses fluorescence only after binding to DNA, the step to remove unbound dye can be omitted. FSC and SSC were used to select mainly neutrophils.

Cells (1 × 10⁵) were seeded in 24-well plates and after 12 hr were transfected with 20 ng/ml of pIC (high molecular weight (1.5–8 kb, InvivoGen) with Lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol. The cells were incubated with 250 nM Sytox-Green dye (Thermo Fisher), a nucleic acid stain that is an indicator of dead cells and which is impermeant to live cells, and 250 nM of cell permeable dye Syto 60-Red (ThermoFisher), which allows quantification of the total number of cells present in each field, using an IncuCyte Live-Cell Imaging System and software (Essen Instruments 2015A) for 36 hr. Cell death was measured by counting the green objects per mm² (dead cells, green) and then normalizing to the total number of cells per mm² (red objects) at each time point using IncuCyte software. The WT and RNASEL KO HME (2 × 10⁵) cells were grown in 12-well plates and transfected with different concentration of pIC (250 ng/ml, 100 ng/ml, 50 ng/ml and 20 ng/ml). Cell death was measured with the IncuCyte system with Sytox-Green dye (250 nM) for 20 hr.

cfDNA in 10 μL platelet-free plasma was measured using an in-house assay, as previously described [14 ]. Briefly, 10 μL of plasma samples from healthy controls or burn patients were added to black opaque Corning 96-well plates in duplicate (Thermo Fisher; Massachusetts, USA) and incubated with 140 μL of SYTOX™ green dye (Thermo Fisher) at a working concentration of 1 μM (diluted in PBS) for 10 min in the dark. For calibration a DNA standard curve (0–1000 ng/mL) of λ-DNA (Thermo Fisher) was included in each assay. Healthy control plasma was added to each plate as a negative control. All samples were run in duplicate. Fluorescence was measured using a BioTek Synergy 2 fluorometric plate reader (NorthStar Scientific Ltd; Potton, UK) with excitation and emission wavelengths set at 485 nm and 528 nm, respectively.

All assays were performed in a 96-well plate format and analysed using a Synergy 2 microplate reader (BioTek). For detection of Annexin V signal, RealTime-Glo Annexin V Apoptosis assay (JA1001, Promega) was used according to the manufacturer’s instructions. For detection of SYTOX Green cell death signal, cells were continuously incubated with a cell-impermeable SYTOX Green dye (2.5 μM, S7020, Thermo Fischer), whose fluorescent intensity was measured at 485/530 nm as previously described [86 (link)]. For detection of caspase-3 activity, cells were continuously incubated with Ac-DEVD-AMC (10 μM, HY-P1003, Medchem Express), whose fluorescent intensity was measured at 330/460 nm as previously described [86 (link)]. Both SYTOX Green cell death signal and caspase-3 activity were normalised to the total DNA fluorescence [87 (link)] measured at 330/460 nm by incubating cells for 30 min with Hoechst 33342 (10 μg/ml in PBS, H3570, Thermo Fischer). For all three cell death assays, data were expressed as a fold change over the non-treated control group.