Polytron homogenizer pt 1200 e

Concentrations of inflammation markers, oxidative stress, and fibrosis were determined in 10% (weight/volume) homogenates of the right lung tissue, which were prepared using 0.1 M ice-cold phosphate buffer (PBS, pH 7.4) and homogenized 5-times for 25 s at 1200 rpm (Polytron homogenizer PT 1200 E, Kinematica AG, Malters, 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.
The ELISA methods was used for measurements of concentrations of below-listed parameters: TNFα (ab236712, Abcam, Cambridge, UK), IL-1β (ab255730, Abcam, Cambridge, UK), IL-6 (ab234570, Abcam, Cambridge, UK); HO-1 (ER1041, FineTest, Wuhan Fine Biotech Co., Ltd., Wuhan, China), hydroxyproline (MAK357, Sigma Aldrich, USA), TGF-β1 (ER1378, FineTest, Wuhan Fine Biotech Co., Ltd., Wuhan, China), NLRP3 (ER1965, FineTest, Wuhan Fine Biotech Co., Ltd., Wuhan, China); catalase, SOD, TAC, and 3-nitrotyrosine (Catalase Activity assay kit, Superoxide Dismutase Activity Assay, Total antioxidant capacity assay, Nitrotyrosine assay, OxiSelect, Cell Biolabs, Inc., San Diego, CA, USA). The manufacturers’ protocols were strictly followed during the measurements and all ELISA analyses were performed in duplicates.

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).