Tnf α elisa kit

Folin–ciocalteau reagent, 4-hydroxycinnamic acid (4-HCA), catechin, quercetin (QR), butylated hydroxytoluene (BHT), 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS), 2′,7′-dichlorofluorescin diacetate (DCFH-DA) probe, HC, thiobarbituric acid (TBA), reduced glutathione (GSH), propidium iodide (PI), collagenase type I, 3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide (MTT), and ALD drug were obtained from Sigma-Aldrich (St. Louis, MO, USA). Roswell Park Memorial Institute (RPMI)-1640 medium, Dulbecco's Modified Eagle Medium (DMEM), α-Minimum Essential Medium Eagle (MEM), trypsin and fetal bovine serum (FBS) were purchased from Lonza, USA. Gene JET RNA purification kit, cDNA synthesis kit, 2X SYBR green master mix kit and protease inhibitor cocktails (PIC) were obtained from ThermoScientific, USA. Osteocalcin ELISA Kit was supplied from Nordic Bioscience Diagnostics (N-MID, Denmark). ALP colorimetric kit was purchased from Biosystems (Barcelona, Spain). The phosphorous colorimetric kit was obtained from Chema Diagnostica (Campania, Italy). TNF-α ELISA kit was purchased from RayBiotech, USA and collagen type IV (COL 4) ELISA kit was supplied from Kamiya Biomedical, US. Primers for NF-κB and COX-2 were purchased from Bioneer, Korea. Other chemicals were obtained with a high grade.

All the chemicals were procured from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India) unless otherwise indicated. Silymarin was obtained from Sigma-Aldrich (USA). Fetal bovine serum (FBS), RPMI-1640, antibiotics and EZcount™ MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Cell Assay Kit were purchased from HiMedia Laboratories Pvt. Ltd. (Mumbai, India). Albumin, γ-glutamyl transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), bilirubin, protein, aspartate transaminase (AST), acid phosphatase (ACP), alanine transaminase (ALT), glucose, urea and cholesterol estimation kits were obtained from Crest Biosystems (Goa, India). TNF-α ELISA kit was procured from Ray Bio (Georgia, United States) and Thiobarbituric acid reactive substances (TBARS) assay kit was purchased from Cayman chemical company (USA). Milli-Q ultrapure water from the departmental central facility was used in the experiments.

TNFα was measured as described by the commercial TNFα ELISA kit purchased from RayBiotech, USA. The TNFα level was determined by the enzyme-linked immunosorbent assay (ELISA) using the anti-rat TNFα precoated microplates (12 × 8 microwell strips).

TNF-α released in culture supernatants of the experimental mice were measured using TNF-α ELISA kit (Ray Bio, USA) according to the manufacturer’s instructions. Absorbance of the sample was immediately measured after the assay at 450 nm using Bio-Rad iMark™ microplate absorbance reader.