Human myeloperoxidase quantikine elisa kit

MPO levels were quantified using the commercially available Human Myeloperoxidase Quantikine ELISA Kit (R&D Systems) and NE levels were assessed by the commercially available Human PMN (Neutrophil) Elastase ELISA Kit (Invitrogen) according to the manufacturer’s instructions.
The quantification of citrullinated histone H3 (CitH3) levels in human plasma was based on the Cell Death Detection Kit (Roche), adjusted in accordance with a published protocol from Thålin et al.^{19 (link)}

ADAMTS13 activity was measured using FRETS-VWF73 (Anaspec Inc; Cat. #AS-63728-01; Fremont, USA). Total ADAMTS13 antigen in plasma was quantified using human ADAMTS13 Quantikine ELSIA kit (R&D Systems; Cat. #DADT130; Minneapolis, USA). VWF antigen was quantified using human VWF-A2 DuoSet ELISA (R&D Systems; Cat. #DY2764-05). Protein C antigen was quantified by ELISA (Affinity Biologicals Inc; Cat. #PC-EIA; Ancaster, Canada). MPO levels were quantified using human Myeloperoxidase Quantikine ELISA kit (R&D Systems; Cat. #DMYE00B). All commercial assays were conducted according to manufacturer’s protocols. ADAMTS13 activity, ADAMTS13 antigen, VWF antigen, PC antigen, and MPO values were expressed relative to healthy control plasma. All parameters were measured on Day 1 and Day Last (non-survivors septic: 10.6±8.8 days, survivors septic: 5.7±3.0 days, and non-septic: 5.0±2.2 days).

Isolated neutrophils were suspended in RPMI 1640 medium supplemented with 10% FCS at 1 × 10⁶ cells/ml and stimulated with 50% apoptotic tumour cell-CM at 37 °C. Neutrophils were collected after 2 or 24 h for flow cytometric analysis of cell surface markers and viability, respectively. Metabolic activity of neutrophils was assessed using the EZ4U Cell Proliferation and Cytotoxicity Assay (BI-5000, Biomedica, Vienna, Austria). Briefly, 5 × 10⁴ neutrophils were cultured in 96-well plates in the presence or absence of 50% apoptotic tumour cell-CM for 24 h, followed by the addition of the EZ4U substrate according to manufacturer’s instructions. After 4 h of incubation at 37 °C, optical densities were analysed on a Varioskan LUX plate reader (Thermo Fisher Scientific). To investigate myeloperoxidase (MPO) release, 1 × 10⁵ neutrophils were suspended in 150 µl Hanks’ Balanced Salt Solution (14025-050, Gibco) and stimulated with an equal volume of FCS-free apoptotic tumour cell-CM for one hour at 37 °C. Neutrophils treated with FCS-free culture medium or the calcium ionophore A23187 (C7522, Sigma) (4 µM) served as negative and positive control, respectively. Supernatants were collected by centrifugation at 450 × g for 10 min and analysed using the Human Myeloperoxidase Quantikine ELISA Kit (DMYE00B, R&D Systems, Abingdon, UK) according to manufacturer’s specifications.

MPO levels from cell supernatant were measured by Human Myeloperoxidase Quantikine ELISA Kit(R&D)according to the manufacturer’s instructions. Add 50 μl sample and 100 μl Assay Diluent to each well. Cover with a plate sealer, and incubate at room temperature for 2 h on a horizontal orbital microplate shaker. Aspirate each well and wash× 4. Add 200 μl of Conjugate to each well, and incubate at room temperature for 2 h on the shaker. Aspirate each well and wash× 4. Add 200 μl Substrate Solution to each well, and incubate at room temperature for 20 min, protect from light. Add 50 μL of Stop Solution to each well. Read at 450 nm within 30 min, and set wavelength correction to 540 nm.