Recombinant human tnf α

ELISA was performed according to the manufacturer’s protocol. Immuno™ Maxisorp™ plates (NUNC-Thermo Fisher Scientific, 442404) were coated overnight at 4°C with anti-human IL-6 (eBioscience™), anti-human TNF-α antibodies (BD Pharmingen™) or anti-human IL-1β antibodies (R&D Systems). Plates were washed and blocked with phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) for 2h at 37°C. Plates were washed and incubated with supernatant or standard dilutions of recombinant human TNF-α (BD Pharmingen™), IL-6 (Thermo Fisher Scientific) or IL-1β (R&D Systems). Biotin Mouse anti-Human TNF-α detection antibody (BD Pharmingen™), Biotin Mouse anti-human IL-6 detection antibody (eBioscience™) or Biotin mouse anti-Human IL-1β detection antibody (R&D Systems) was added as secondary antibody. RPMI medium was used as a negative control. Serum from LPS-activated WB was used as a positive control. Plates were washed and incubated with 3,3′,5,5′-tetramethylbenzidine (TMB) after which the reaction was stopped with sulfuric acid (0.8 M H₂SO₄). The absorbance was measured at OD450 nm on a SpectraMax^® iD3 Reader with SoftMax Pro 7 software. All conditions were performed in duplicate.

Hyclone (Logan, UT, USA) provided Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 medium, and fetal bovine serum (FBS). We obtained EBM-2 medium and EGM-2 Endothelial SingleQuots Kit from Lonza (Basel, Switzerland) and 2-mercaptoethanol from Gibco (Grand Island, NY, USA). We purchased recombinant human TNF-α from BD Pharmingen (San Diego, CA, USA) and recombinant mouse TNF-α from NKMAX (Seongnam-si, Gyeonggi-do, Korea). Sigma-Aldrich (St. Louis, MO, USA) provided 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and analytical-grade reference standards (methyl gallate, catechin hydrate, paeoniflorin, benzoic acid, and paeonol). We used horseradish peroxidase (HRP)-conjugated pierce goat antirabbit IgG (H+L), pierce goat antimouse IgG (H+L), and Alexa Fluor 488 and 594 goat antirabbit IgG (H+L) for the secondary antibodies, purchasing these from Thermo Fisher Scientific Inc. (Eugene, OR, USA). The PLE reference standards, oxypaeoflorin, benzoyl paeoniflorin, and albiflorin, were purchased from Ensol Biosciences Inc. (Daejeon, Korea), Biosynth Carbosynth (San Diego, CA, USA), and FUJIFILM Wako Pure Chemical Corporation (Richmond, VA, USA).