Tnf α elisa kit

SBKT, isolated from the pulp, was obtained from Food and Herbal Park, Patanjali Ayurveda Limited, Haridwar, India. Culture media RPMI-1640, fetal bovine serum, and antibiotic/anti-mycotic mixture were obtained from Gibco. Cytokines interleukin 1-beta (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) ELISA kits were purchased from BD Biosciences. LPS, TPA, λ-Carrageenan, indomethacin (INDO), and dexamethasone (DEXA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hematoxylin, potassium aluminium sulfate dodecahydrate, and mercury (II) oxide red were purchased from Merck India Pvt. Ltd, Mumbai, India. Eosin yellow and ferric chloride were purchased from Hi-Media Laboratories, Mumbai, India. All the other chemicals and reagents purchased for the study were of the highest commercial grade.

Mouse C2C12 myoblasts were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Dulbecco’s phosphate buffered saline (D-PBS), horse serum, penicillin, and streptomycin were from GIBCO (Grand Island, NY). Zileuton and LTB4 were from Tocris (Bristol, UK), and AmplexRed from Invitrogen (Waltham, MA). A769662 was obtained from LC laboratories (Woburn, MA). Antibodies were obtained as follows: GRP78, ATF3, CHOP, Akt and pAkt (Ser473) (Thr307) from Santa Cruz Biotechnology (Santa Cruz, CA); AMPK, pAMPK, pACC (Ser79) and ACC from Cell Signaling Technology (Danvers, MA); pIRS-1 (Ser307) and IRS-1 from Bioworld (Minneapolis, MN); 5-LO from Novusbio (Littleton, CO) and β-actin from Sigma-Aldrich (St. Louis, MO). 5-LO siRNA (sc-29597), AMPK1/2 siRNA (sc-45313) and control siRNA (sc-37007) were from Santa Cruz Biotechnology (Santa Cruz, CA). Oligonucleotide primers were from Bioneer Co. Ltd. (Daejeon, Korea). LTB4 kits were from Enzo Life Sciences (Seoul, Korea) and MyBioSource, Inc. (San Diego, CA). CysLTs kit and U75032 were from Cayman chemicals (Ann Arbor, MI). IL-6 and TNF-α ELISA kits were from BD Biosciences (San Diego, CA). All other reagents were from Sigma Chemicals (St. Louis, MO) unless indicated otherwise.

Carvacrol (≥98%, food-grade), Dulbecco’s phosphate-buffered saline (DPBS), dimethyl sulfoxide (DMSO), lipoteichoic acid (LTA), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and phenazine methosulfate (PMS) were obtained from Sigma-Aldrich (Oakville, ON, Canada). Peptidoglycan (PGN) was obtained from Cedarlane Laboratories (Burlington, ON, Canada). Human tonsil epithelial cells (HTonEpiCs), tonsil epithelial cell medium, tonsil epithelium cell growth supplement, poly-L-lysine (PLL), trypsin neutralization solution (TNS), and trypsin-ethylenediamine tetra acetic acid (0.25%) solution (TE) were purchased from ScienCell Research Laboratory (San Diego, CA, USA). Fetal bovine serum (FBS) was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). 7-Aminoactinomycin D (7-AAD) stain was purchased from BioLegend, Inc. (San Diego, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from different manufacturers: IL-6 and TNF-α ELISA kits were purchased from BD Biosciences (Mississauga, ON, Canada); human ENA-78, GCP-2, and human COX-2 ELISA kits were purchased from RayBiotech, Inc. (Norcross, GA, USA); a human BD-2 ELISA kit was purchased from PromoCell GmbH (Sickingenstraße, Heidelberg, Germany); and PGE₂ and IL-8 kits were purchased from Invitrogen (Nepean, ON, Canada).

CBD and THCV were purchased from ChemFaces (Wuhan, Hubei, PRC), while CBN was purchased from Cayman Chemical (Ann Arbor, MI). All pCBs were diluted in absolute ethanol. The final concentration of absolute ethanol in the culture medium was 1:1000.
Fetal Bovine Serum (FBS, Gibco™), and cell culture media (RPMI1640) were purchased from ThermoFisher Scientific (Waltham, MA, USA). IL-6, IL-10, IL-12, CXCL8 and TNFα ELISA kits were all from BD-Biosciences (San Jose, CA, USA). LPS and CL075 was obtained from Invivogen (San Diego, CA, USA) and dissolved in nuclease-free water.
Fluorescently labeled monoclonal antibodies (mAbs) against DC-SIGN (RRID: AB_1134045), CD83 (RRID: AB_314514), CD1a (RRID: AB_314020), CD207 (RRID: AB_2561590), CD4 (RRID : AB_571945) were sourced from BioLegend (San Diego, CA, USA), HLA-DQ (RRID: AB_2573320) mAbs were obtained from ThermoFisher Scientific, while CD86 (RRID: AB_2275742), IL4 (RRID : AB_357279) and CCR7 (RRID: AB_2259847) were from R&D Systems (Minneapolis, MI, USA), and CD3 (RRID : AB_395736) was from BD-Biosciences.

The effect of ME-3^® on inflammatory cytokine release, including TNF-α (ELISA kit; BD Bioscience, Franklin_Lakes, NJ, USA), IL-6, and IL-8 (ELISA kit; Mabtech, Sigma-Aldrich), was evaluated using the Caco-2/U937 co-culture described above in Model b. In this case, dU937 cells were pretreated with ME-3^® and then stimulated with LPS.
The cytokines released in the medium from the apical or basolateral chamber were detected using the ELISA kits according to the manufacturer’s instructions. The control group was represented by dU937 not stimulated with LPS.