Rat il 6 quantikine elisa kit

Cell supernatants were collected and tested for IL-6 using a rat IL-6 Quantikine^® ELISA kit (#R6000B, R&D Systems) or TNF-α using a rat TNF-α Quantikine^® ELISA kit (#RTA00, R&D Systems). Plates were read on a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA, USA) and analyzed using SOFTmax analysis software (Molecular Devices).

Plasma samples were used to monitor the plasma concentration of IL-6, glucose, LDL, HDL, and PSCK9. The following ELISA were used to quantify these samples: IL-6 (Rat IL-6 Quantikine ELISA Kit, R&D Systems, USA/R6000B), glucose (Rat Glucose Assay Kit, Crystal Chem, USA/81693), LDL (Rat LDL-Cholesterol Assay Kit, Crystal Chem, USA/79960), HDL (Rat HDL-Cholesterol Assay Kit, Crystal Chem, USA/79970) and PSCK9 (Rat Proprotein convertase subtilisin/kexin type 9(PCSK9) ELISA kit, Cusabio, USA/CSB-EL017647RA).

For the detection of SASP factors in the serum (n = 4) or serum‐free conditioned culture medium of isolated HSC (n = 3) from young and old rats, the Proteome Profiler Rat Cytokine Array Kit (R&D Systems) was used according to the manufacturer's instructions. Analysis of serum samples revealed that 10 out of 29 factors showed a reaction on the membrane while only two factors could be detected on the membrane incubated with the culture medium. Visualization and documentation were performed with ChemiDoc Imaging System (BioRad) and protein dot intensities were analyzed with the AlphaView software (Proteinsimple). Furthermore, culture supernatants of HSC from young (n = 5) and old (n = 3) rats were analyzed with the Rat CXCL3/CINC‐2 alpha/beta Quantikine ELISA Kit (RCN200, R&D Systems) and the Rat IL6 Quantikine ELISA Kit (R6000B, R&D Systems) according to manufacturer's recommendations. Data from the cytokine array and ELISA analyses were normalized to cell number per area, which was assessed by counting the cells with the cellSens Dimension 1.16 software at three different positions of each culture dish (Olympus). To investigate the amount of HGF released by HSC after shear stress (n = 5) and stretching (n = 4) as well as by culture on different ECM proteins, culture supernatants were analyzed with the Mouse/Rat HGF Quantikine ELISA (R&D Systems).

Low-glucose DMEM, foetal bovine serum (FBS), Trypsin and HBSS were from Gibco Pasadena, CA (USA). Qiliqiangxin extracts were provided by Shijiazhuang Yiling Pharmaceutical Co., Shijiazhuang Ltd (China); Olmesartan (OLM) was purchased from Shanghai Sankyo Pharmaceutical Co., Shanghai Ltd. (China); Ang II was purchased from Sigma-Aldrich Co., St. Louis, Missouri LLC. (USA); Rabbit anti-rat α-SMA polyclonal antibody was from Bioworld Technology, Nanjing Inc. (China); Rabbit anti-rat TGF-β₁ polyclonal antibody was from Santa Cruz Dallas, Texas (USA). Rat IL-6 Quantikine ELISA Kit was from R&D Systems, Minneapolis, MN Inc (USA), IL-6 RNA lentivirus vector was from Shanghai GeneChem Co., Shanghai Ltd (China).

Two proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), had been assessed in the liver tissues. TNF-α levels were measured using a commercially accessible enzyme-linked immunosorbent assay (ELISA) kits (catalog number: CSB-E11987r, Cusabio Biotech Co., Ltd.) following the manufacturer's protocol. Meanwhile, the concentration of IL-6 had been assessed using the Rat IL-6 Quantikine ELISA Kit subsequent to the instructions described by the manufacturer (R&D Systems, Inc., Minneapolis, USA).