Myeloperoxidase mpo

Neutrophils (5 × 10⁶/mL) were pre-incubated for 10 min with APPA (100 µg/mL), before priming with GM-CSF (5 ng/mL) for 30 min and then stimulated to degranulate with cytochalasin B (5 µg/mL) plus fMLP (1 µM, both from Sigma) for 15 min. Cells were pelleted gently, washed and analysed by flow cytometry; while, supernatants were removed for SDS-PAGE after adding concentrated Laemmli protein sample buffer. After electrophoresis, proteins were transferred to PVDF membranes and probed with antibodies to myeloperoxidase (MPO) (R&D Systems), lactoferrin (Abcam), MMP9 (R&D Systems) and elastase (Abcam). Secondary antibodies were anti-rabbit IgG (GE Healthcare) and anti-mouse IgG (Sigma) HRP-linked antibodies (1:10,000). Bound antibodies were detected using the ECL system (Merck) and film (Amersham).

Murine tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), chemokine C-X-C motif ligand 1 (CXCL1), amphiregulin (AREG), granulocyte-macrophage colony-stimulating factor (GM-CSF), myeloperoxidase (MPO), monocyte chemoattractant protein-1 (MCP-1), and macrophage migration inhibitory factor (MIF), Duoset ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). RvD1 and RvD2 murine ELISA kits were purchased from Cayman Chemicals (Ann Arbor, MI, USA). DHA and standard mouse chow diets were prepared by Envigo (Madison, WI, USA) as previously described [30 (link)].

Tumor homogenate was prepared for in vivo viral titer assessment as described above. The homogenate supernatant was used for ELISA and western blot assay. TNF-α (eBioscience, San Diego, CA) and IFN-β (PBL Assay Science, Township, NJ) ELISA was performed according to manufacturer's instructions. H₂O₂ measurement was performed using a hydrogen peroxide chemiluminescent kit (Enzo Life Sciences, Farmingdale, NY) following manufacturer's instructions. ELISA and H₂O₂ data were corrected for tumor weight by dividing concentration by total tumor weight. Western blot was performed as previously described [15 (link)]. Briefly, 4x LDS buffer (Invitrogen, Carlsbad, CA) containing β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO) was added to 1x and 5% (vol/vol) concentrations, respectively and gel electrophoresis using the Novex (Invitrogen, Carlsbad, CA) western blotting system was performed. A Licor Odyssey Fc imager (Licor, Lincoln, NE) was used to image immunoblots. Antibodies used for immunoblot recognized Nitric oxide synthase (NOS), Stat1-p (Y701), Stat1 (Cell Signaling Technologies, Danvers, MA); Tubulin (Sigma-Aldrich, St. Louis, MO); and myeloperoxidase (MPO; R&D Biosystems, Minneapolis, MN).

Inflammatory cytokine levels in BALF and culture media were measured by ELISA. RAW 264.7 cells (5 × 10⁵ cells/well in a 12-well plate) were pretreated with EELCT (1, 10, and 100 μg/mL) and Dex (10 μM) for 1 h and stimulated with LPS (100 ng/mL) for 12 h or 24 h. ELISA was performed on a 96-well immune plate (Nunc, Rochester, NY) using specific kits according to the manufacturer's instructions. The following kits were used: TNF-α and IL-6 (BD Biosciences, San Diego, CA); IL-1β (Invitrogen, Carlsbad, CA); and myeloperoxidase (MPO) (R&D Systems, Minneapolis, MN). The absorbance was measured at 450 nm using a spectrophotometer (Molecular Devices).

Male specific-pathogen-free (SPF) Sprague-Dawley rats, weighing 200 ± 20 g, were provided by Animal Center of Anhui Province. Animals were allowed one week of adaptation before entering the experimental period. Rats were housed at room temperature with a 12 h–12 h light–dark cycle. TNBS (031M5021), DL-Hcy (MW 135.18, 121M39044) and Evans Blue (MW 960.8) were purchased from Sigma (USA). Interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), myeloperoxidase (MPO) and malondialdehyde (MDA) test kits were obtained from R&D (USA). Matrix metalloproteinase-2,9 (MMP-2,9) test kits were purchased from Wuhan Xinqidi Biological Technology Co., Ltd. (China).