Elisa kit for pge2

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and various other tissue culture reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Primary antibodies are anti-iNOS, sc-650; anti-COX-2, sc-1745; anti-IκB-α, sc-371; anti-p-IκB-α, sc-8404; anti-p50, sc-7178; anti-p65, sc-8008, from Santa Cruz Biotechnology (Dallas, TX, USA) anti-p-ERK, #9101; anti-ERK, #9102; anti-p-JNK, #9251; anti-JNK, #9252S; anti-p-p38, #9211; anti-p38, #9212S, from Cell Signaling Technology (Danvers, MA, USA) Secondary antibodies: anti-mouse, ap124p; anti-goat, ap106p; anti-rabbit, ap132p, Millipore. The enzyme-linked immunosorbent assay (ELISA) kit for PGE₂ was purchased from R&D Systems (Minneapolis, MN, USA). The compounds glabridin, HSG4112, (S)-HSG4112, HGR4113 were provided by Glaceum Inc (Suwon, Republic of Korea).

RAW 264.7 cells (2 × 10⁵ cells/mL) were seeded onto a 24-well plate and then incubated with/without LPS (100 ng/mL) in the presence or absence of SAMPLE (1, 10, 100 nM and 1, 10 μM) for 24 h. Nitrite levels of cellular supernatants were measured using the Griess reaction and estimated to reflect the concentration of NO. The absorbance was measured at the wavelength of 540 nm using the microplate reader. In all experiments, fresh culture media were used as blanks. The levels of nitrite in the samples were determined using the standard curve of sodium nitrite. PGE₂ concentration in the medium was measured using an ELISA kit for PGE₂ (R&D Systems, Minnesota, MN, USA).