MPO levels in the vitreous samples 100 µl/well (the samples were diluted 1:200 with a dilution buffer, R&D Systems) were measured using the Human MPO DuoSet enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer’s protocol (R&D Systems). MPO is present in extruded NET complexes mostly associated with H3Cit and NE. Such complexes were quantified with modified capture ELISA [11 (link)] In brief, a 96-well plate was precoated with human anti-H3Cit (Abcam, dilution 1:12,000) or human anti-NE antibodies (Abcam, dilution 1:12,000) overnight. The following day vitreous sample (diluted 1:200 with a dilution buffer, from the MPO DuoSet ELISA kit, R&D Systems) after which the MPO-associated complexes were detected using the human MPO ELISA kit, according to the manufacturer’s protocol (R&D Systems).
Human mpo elisa kit
Manufactured by R&D Systems
Sourced in United States
The Human MPO ELISA Kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of human myeloperoxidase (MPO) levels in biological samples such as plasma, serum, and cell culture supernatants.
Automatically generated - may contain errors
4 protocols using human mpo elisa kit
1
Quantification of Vitreous MPO and NET Complexes
2
Quantifying MPO, HNE, and NET in Plasma
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Concentrations of MPO and HNE in human plasma were measured by commercial ELISA kits (Human MPO ELISA kit, R&D Systems, Minneapolis, MS, USA; Human PMN elastase ELISA kit, Abcam, Cambridge, MA, USA) following manufacturers’ instructions. Levels of MPO-DNA complexes were assessed by an MPO-DNA ELISA as described previously [12 (link), 13 (link)]. Briefly, human plasma samples were diluted 10-fold in sterile PBS and subjected to MPO-DNA ELISA. Results are expressed as percentages of the “NET-standard” that contains pooled supernatants of PMA-stimulated human neutrophils and serves as a reference [13 (link)].
3
Quantification of Inflammatory Biomarkers
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Blood samples of humans or rats containing the anticoagulant EDTA were centrifuged at 3000 g for 10 min at room temperature, and then the upper two-thirds of the plasma were carefully removed for testing. The samples were centrifuged at 500 g for 5 min, and the upper two-thirds of the cell culture supernatant were carefully aspirated for testing. The experimental steps were performed according to the instructions of the corresponding ELISA kit. The following kits were used: human HBP ELISA kit (Thermo Fisher, USA), human MPO ELISA Kit (R&D Systems, USA), human elastase ELISA kit (R&D Systems, USA), human MMP9 ELISA kit (R&D Systems, USA), human hyaluronic acid ELISA kit (Biorbyt, UK), human heparan sulfate ELISA kit (Biorbyt, UK), human SDC-1 ELISA kit (Biorbyt, UK), rat HA ELISA kit (Cusabio, China), rat HS ELISA kit (Cusabio, China), and rat SDC-1 ELISA kit (Cusabio, China).
4
Quantifying Airway NET Biomarkers
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To quantify NETs from the airway fluid, a capture ELISA myeloperoxidase (MPO) associated with DNA was performed as previously described15 (link). For the capture antibody, Human MPO ELISA kit (R&D Systems, Minneapolis, MN) was used according to the manufacturer’s instructions. We added 200 µL of airway fluid to the wells and incubated for 1 h. After washing 3 times with 300 µL of wash buffer, 100 µL incubation buffer containing a peroxidase-labeled anti-DNA mAb (component 2, Cell Death ELISAPLUS, Roche) was used and expressed as relative fluorescence units (RFU). Each sample’s RFU value was normalized to the mean and standard deviation (SD) for each experiment using the standardization formula: (1 + (sample value − mean experiment value)/SD experiment value) for each experiment.
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