Kge001

Human oxidised LDL in serum was determined by OxiSelect™ Human Oxidized LDL ELISA kit (OxPL-LDL Quantification, Cell Biolabs, San Diego, CA, U.S.A.) in serum samples collected from patients and controls as described above. The method quantifies the level of oxidised phospholipids associated with human LDL, and the measurement was performed according to the manufacturer’s recommendations. Total antioxidant capacity (PAO test kit; KPA-050, JaICA, Shizuoka, Japan), and the total nitric oxide metabolites; nitrite and nitrate (KGE001; R&D systems, Minneapolis, MN) were measured in serum according to the manufacturers’ recommendations. Absorbance was measured at 450 nm for oxidised LDL and 480 nm for total antioxidants by using a Multiskan Ascent microplate reader (Thermo Labsystem, Stockholm, Sweden), and NO metabolites at 540 nm with correction at 690 nm by using Spectramax 190 plate reader (Molecular Devices, Sunnyvale, CA).

Circulating NO blood levels were tested using ELISA commercial kit (#KGE001, R&D). The method is a spectrophotometric that uses Greiss reaction which is based on enzymatic conversion of nitrates to nitrites due to nitrate reductase [27 (link), 28 (link)].

All analyses were performed at the Institute of Pharmacology and Toxicology of the RWTH Aachen (Aachen, Germany). For the parameters IL-1β, IL-6, IL-8, IP-10 and MIP-1α a Bio-Plex Cytokine assay (Bio-Rad Laboratories, Munich Germany) was used [55 (link)]. Enzyme-linked immunosorbent assays (ELISA) were used for albumin (# EA2201-1; AssayPro; St. Charles, USA), nitrotyrosine (# HK501; Cell Sciences; Canton, USA), and TGF-β₁ (# DB100B; R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany). Furthermore, competitive binding assays were employed for LTB₄ (# KGE006B; R&D Systems GmbH) and NPY (# EK-049-03; Phoenix Europe GmbH, Karlsruhe, Germany). Nitrite concentration was analyzed using a Griess reaction assay (# KGE001; R&D Systems GmbH). All assays were performed according to the manufacturer’s recommendations. For ASM, a proprietary assay was used [56 (link)]. Samples from the same patient and day were pooled to increase the amount and decrease variations of dilution. No attempt to normalize data was made since no uniformly accepted standard is currently available. We expressed the data per milliliter of TA as recommended by the European Respiratory Task Force on Bronchoalveolar Lavage in children [57 (link),58 (link)] and reported by others [59 (link),60 (link)].

Blood samples were taken from the right atrium, hepatic vein, and portal vein before insertion of the TIPS stent and 7 days after the TIPS procedure (Figures 1 and 2). Plasma samples were centrifuged at 1800 g for 15 min at 4°C and immediately stored at −80°C until they were analyzed. Serum LPS and ET-1 were measured by enzyme linked immunosorbent assay (ELISA) as previously described [6 (link)]. Control samples and serum standards were jointly analyzed in each run. The interassay coefficient of variation in the current study (six runs) was ~10%. Serum NO was measured from the nitrate/nitrite content using a fluorometric assay (KGE 001; R&D Systems China, Shanghai, China). All other analyses were performed using standard laboratory methods.

In order to study the nitric oxide (NOX) release in supernatants, we measured nitric oxide metabolites of nitrite and nitrate using a specific assay kit from R&D Systems (catalog number KGE001) according to the manufacturer's instructions. Results were expressed in μmol/L. Total RNA was quantified as described above for iNOS (inducible NOS), eNOS (endothelial NOS) and COX2 quantification. Each real‐time PCR was carried out in triplicate in a total of 20 μL reaction mixture. Primers used for real‐time PCR analysis were purchased from Life Technologies. The housekeeping gene, glyceraldehyde (Kimmel et al., 2016)phosphate dehydrogenase (GADPH), was amplified in each sample as control and was used for normalization. Data analysis was performed using the ΔΔCt method.

To obtain the conditioned media for cytokine array analysis, NP cells were subjected to different treatments (Control, IL-1β, IL-1β+SRR) for 24 hours without changing the culture medium. Subsequently, the conditioned media from each group were collected and centrifuged at 5000 rpm and 4°C for 15 minutes. The supernatants were stored at -80°C separately until use and were diluted with appropriate standard diluents before measurement. The levels of ADAMTS5 (DY2198-05, R&D Systems, USA), collagen-II (F15215, Westang, China), MMP-3 (F01870, Westang, China), Aggrecan (DY1220, R&D Systems, USA), MDA (F01963, Westang, China), SOD1 (F11502, Westang, China), TNF-α (F02810, Westang, China), IL-6 (F01310, Westang, China), Nitrite (KGE001, R&D Systems, USA), and PGE2 (F02290, Westang, China) in the supernatants were determined using Enzyme-Linked Immunosorbent Assay (ELISA) kits. The optical density was measured at a wavelength of 450 nm.