Cell death detection elisa

Nucleosomes were measured in plasma using Cell Death Detection ELISA (Roche). A standard curve was prepared by making serial dilutions of a DNA-histone complex of a known concentration, supplied by the manufacturers. MPO levels were determined by LEGEND MAX™ ELISA (Biolegend) and histone-MPO complexes by ELISA, as described50 (link), with modifications. Briefly, biotinylated anti-histone antibody (1:25 dilution) was added to streptavidin-coated plates (Cell Death Detection) and incubated during 2 hours. After blocking with 2.5% bovine serum albumin, plasma samples (1:4 dilution) were incubated during 2 hours followed by 2-hour incubation with HRP-conjugated anti-MPO antibody (Quantikine ELISA Human MPO, R&D, Minneapolis, MN, USA) and addition of tetramethylbenzidine, all at room temperature. Identical procedure was carried out for each sample omitting the anti-histone antibody. Absorbance at 450-nm wavelength of samples without anti-histone antibody (non-specific signal) was substracted from that of duplicate samples with anti-histone antibody (histone-MPO complexes). Supernatant of PMA-stimulated and unstimulated neutrophils was used as positive and negative control for NETosis, respectively. Antigen levels of VWF were measured by ELISA, as described51 (link) and D-dimer by immunoturbidimetric assay (Innovance D-dimer, Siemens, Erlanger, Germany).

Cell death detection ELISA (Roche Applied Science, Germany), determinates the mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates after induced cell death using mouse monoclonal antibodies against DNA and histones.^{34 (link)} Briefly, cells were incubated with the IC50 concentrations of methanolic and dichloromethane extract S. atropatana. DMSO and Taxol were used as negative and positive controls respectively. After 24 hours of incubation at 37°C, the culture supernatants were utilized for quantification of necrosis and cell lysates for apoptosis. The assay was performed according to the manufacturer’s instructions. The absorbance was measured using an ELISA plate reader at 405 nm and the percentage of apoptosis and necrosis obtained from the ratio of absorbance in the treated samples to that of the untreated controls.

Human neutrophils (4 × 10⁵/100 μl) were incubated with SARS‐CoV2‐spike Pseudotyped virus (WT, MOI = 0.1) in present autologous platelets (4 × 10⁶/100 μl) for 3 h at 37°C. Supernatant was harvested by centrifugation at 500 g for 5 min at room temperature. 96‐well Clear Flat Bottom Polystyrene High Bind Microplate (#9018, Corning) was coated with 50 μl of rabbit anti‐MPO serum (#07‐496, Millipore; dilution 1:1,000) and rabbit anti‐elastase polyclonal antibody (# sc‐25621, Santa Cruz; clone H‐57; 2 μg/ml) at 4°C overnight (14‐16 h). Plate was washed twice with PBS and then incubated with 50 μl of sample for 2 h at room temperature. After washing three times with 200 μl PBST (PBS containing 0.05% Tween 20), add 100 μl of detection antibody (HRP‐conjugated anti‐DNA antibody from Roche #Cell Death Detection ELISA; dilution 1:40) then incubated for 1 h at room temperature. Plate was washed three times with 200 μl PBST and then incubated with 200 μl of ABTS for 10–20 min. Measure the absorbance at 405/490 nm.

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.