Rat il 1 beta elisa kit

TNF-α, IL-1β, NF-𝜅B levels in serum were determined using an ELISA kit and the manufacturer’s instructions. Rat NF kappaB p65 ELISA Kit (ab176648), was used and obtained from Abcam, United states. Rat TNF alpha ELISA kit (ab181421) is a single-wash 90 min sandwich ELISA designed for the quantitative measurement of TNF alpha protein (Abcam, united states), Rat IL-1 beta ELISA Kit (ab255730) is a single-wash 90 min sandwich ELISA designed for the quantitative measurement of IL-1 beta protein in serum, cell culture and plasma. The Lowry Method (1951) [66 (link)] was used to determine the protein levels in samples. For histopathological examination of synovial tissue, a portion of it was fixed in 4% paraformaldehyde buffer. In addition, immunohistochemistry was used to quantify inflammatory markers.
Ohkawa et al. (1979) [67 (link)] described a method for measuring lipid peroxide (Malondialdehyde; MDA). Ellman (1959) [68 (link)] was used to measure the concentration of reduced glutathione (GSH). Glutathione reductase was measured using the method described by Hsiao et al. (2001) [69 (link)]. Fridovich (1989) [70 (link)] described a method for measuring Superoxide Dismutase (SOD) activity. The Sinha (1972) method was used to determine catalase activity [71 ].

The culture supernatants were collected and centrifuged at 250 g for 5 min before storage at − 80 °C. Microglia were lysed using the denaturing M-PER Mammalian Protein Extraction Reagent (78503, Thermo Fisher Scientific) supplemented with the protease inhibitor cocktail (S8820, Sigma-Aldrich) for 5 min on ice. The cell lysates were then centrifuged at 10000g for 5 min before storage at − 80 °C. The indicated cytokines were measured with the Rat IL-6 ELISA Set (550319, BD Biosciences), Rat TNF ELISA Set (558535, BD Biosciences), and Rat IL-1 beta ELISA Kit (ab100768, Abcam), respectively.

The synovial fluid samples of OA rats and the supernatant samples of RAW264.7 cells were collected. Cytokine levels were measured using the following ELISA kits, according to the manufacturer's protocol (All from Abcam, USA): Mouse IL-6 ELISA Kit, Mouse IL-1 beta ELISA Kit, Mouse TNF alpha ELISA Kit, Mouse IL-10 ELISA Kit, Rat IL-6 ELISA Kit, Rat IL-1 beta ELISA Kit, Rat TNF alpha ELISA Kit, and Rat IL-10 ELISA Kit. The results were tested using a multifunctional enzyme marker.

The levels of IL-1β, PGE2, and COX-2 in the cell culture supernatant or serum were determined by Rat IL-1 beta ELISA kit (Abcam, catalog number: ab255730), Rat PGE2 ELISA Kit (Wuhan Fine Biotech Co., Ltd, catalog number: ER1800), ELISA kits, and Rat COX-2 ELISA Kit (CUSABIO TECHNOLOGY, catalog number: CSB-E13399r) according to the manufacturer's instructions. The experiments were performed more than three times independently.

The levels of IL-1β and IL-18 in rat ovaries were measured using the Rat IL-1 beta ELISA Kit (ab255730, Abcam) and Rat IL-18 ELISA Kit (ab213909, Abcam) following the instructions, respectively. Optical density values were determined with a plate reader.

To assess the microglial secretion of TNFα, IL-1β, IL-10 and IFNβ, the culture medium was harvested, spun down at 1000× g for 5 min at 4 °C, frozen on dry ice and stored at −80 °C. On the day of analysis, all reagents were thawed and adjusted to RT before the culture medium was measured in duplets using the following colorimetric sandwich ELISA kits according the manufacturers protocol: rat TNF alpha ELISA Kit (ab100785, Abcam), rat IL-1 beta ELISA kit (ab100768, abcam), rat IL-10 ELISA Kit (ab100764, abcam), Rat IFN-beta ELISA Kit (NBP3-06753, Novus Biologicals). After the generation of a 4-parameter logistic standard curve using Graph Pad Prism 8.4.3 (GraphPad Software, San Diego, CA, USA), total protein concentrations were calculated. Protein levels below the detection limits of the used ELISAs were set to 0.