Oxiselect nitrotyrosine elisa kit

Hyphae of P. infestans (0.2 g) were ground in liquid nitrogen to a fine powder, then it was suspended in buffer containing 50 mM Tris–HCl (pH 7.6) (Bio-Rad; Hercules, CA, USA) with 2 mM EDTA (BioShop), 2 mM DTT (Sigma; Saint-Louis, MO, USA), and 1 mM PMSF (Sigma; Saint-Louis, MO, USA). After centrifugation (10,000 g for 15 min at 4 °C) the supernatant was collected and the protein concentration was determined with the Bradford [56 (link)] assay. 3-nitrotyrosine in a protein sample was measured using the OxiSelect^TM Nitrotyrosine ELISA Kit (Cell Biolabs; San Diego, CA, USA; STA-305) according to the manufacturer’s protocol. Optical density was measured at 450 nm using an IMARK^TM Microplate Reader (Bio-Rad; Hercules, CA, USA). The 3-nitrotyrosine content in protein samples was determined by comparing with the predetermined 3-nitrotyrosine standard curve. Each sample was analyzed in triplicate on ELISA microplates.

Concentrations of cytokines and oxidative modification products were determined in 10% (weight/volume) lung homogenate prepared using 0.1 M ice-cold phosphate buffer (PBS, pH 7.4) by Polytron homogenizer PT 1200 E (5-times for 25 s, 1200 rpm; Kinematica AG, Switzerland). Homogenates were then frozen 3 times and centrifuged (12,000 rpm, 15 min, 4 °C). Final supernatants were then stored at −70 °C until the analysis was performed.
Concentrations of IL-6, IL-1β, IL-10 (USCN Life Science Inc., Wuhan, China), and RAGE (MyBioSource, San Diego, CA, USA) were quantified using rabbit-specific ELISA kits according to the manufacturers’ instructions. Data were expressed in pg/mL.
Protein oxidative damage was determined using OxiSelect^TM Nitrotyrosine ELISA Kit (Cell Biolabs, Inc., San Diego, CA, USA). Data were expressed in 3-nitrotyrosine nanomole concentration (nM, 3NT). Lipid oxidative damage expressed by the concentration of thiobarbituric acid reacting substances (TBARS) was determined by OxiSelect^TM TBARS Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA). Data were expressed as malondialdehyde in micromole concentration (μM MDA).
Total antioxidant capacity (TAC) was determined using an ELISA kit according to the manufacturer’s instructions (Cell Biolabs, Inc., San Diego, CA, USA). Data were expressed as micromole concentration of copper reducing equivalents (μM CRE).