Mouse tnfalpha elisa kit

To perform the experiment RAW 264.7 cells were seeded into a 24-well plate at a density of 5 × 10⁴ cells well⁻¹. To test the effect of the preparations in the case of chronic inflammation, cells were treated with the preparations for 2 h, and then stimulated with 1 µg mL⁻¹ of lipopolysaccharide (LPS) O55: B5 from Escherichia coli for 18 h. Untreated cells and LPS were the negative control of inflammation, and cells treated with LPS without samples were the reference model of inflammation (positive control). After the cells’ treatment with samples for 24 h, the medium was collected and centrifuged at 150× g for 5 min to precipitate the cell debris. In the supernatants the protein concentrations of IL-6 (Mouse IL6 ELISA kit, Biorbyt Ltd., Cambridge, UK) and TNF-α (Mouse TNFalpha ELISA kit, Biorbyt Ltd., Cambridge, UK) were determined using ELISA kits, following the manufacturer’s instructions. The accumulation of NO metabolite in the cell culture supernatant was measured using Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride; Sigma Aldrich), where an equal volume of the supernatant was mixed with Griess reagent in a 96-well plate. After incubation at room temperature and darkness for 15 min, the absorbance was measured at 540 nm.

To perform, the experiment cells were seeded into a 6-well plate at a density of 2 × 10⁵ cells/well. On the last day of cells treatment the medium was collected and protein concentrations of adiponectin (Adiponectin Mouse ELISA Kit, Abcam, Cambridge, GB), leptin (Leptin Mouse ELISA Kit, Abcam, Cambridge, GB), Il6 (Mouse IL6 ELISA kit, Biorbyt Ltd., Cambridge, GB) and TNFα (Mouse TNF alpha ELISA kit, Biorbyt Ltd., Cambridge, GB), were determined using ELISA kits, following the manufacturer’s instructions. The PPARγ protein level present in the nuclear fraction of the cellular lysates was determined with the PPARγ Transcription Factor Assay Kit (Abcam, Cambridge, GB).