3 3 5 5 tetramethylbenzidine (tmb)

Binding between Mac-1 and CD147 was evaluated using a modified enzyme-linked immunosorbent assay (ELISA). In a 96-well plate, wells were coated with recombinant Mac-1 (R&D Systems, Minneapolis, MN, USA) or bovine serum albumin (BSA) as the control. Recombinant CD147 was added in increasing concentrations (0–20 µg/mL) for 1 h. After removing CD147 and gentle washing, the wells were incubated with an anti-CD147 antibody (mouse-anti CD147, Abcam, Cambridge, UK) followed by a biotinylated secondary antibody (polyclonal rabbit anti mouse, Dako, Glostrup, Denmark) and a streptavidin/HRP complex (Life technologies, Carlsbad, CA, USA). The binding was detected using 3,3′,5,5′-tetramethylbenzidine (Serva, Heidelberg, Germany). The reaction was stopped using 1 M H₂SO₄. The absorption was measured using an ELISA plate reader at 450 nm with a reference value of 570 nm. Measurements from 6 independent experiments were analyzed.

Analytical-grade salts, buffer reagents, chloramine T, hydrochloric acid (HCl), acetic acid (CH₃COOH), potassium iodide (KI), methanesulfonic acid (CH₄O₃S), adrenaline, glutathione reductase (GR), peroxidase, and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich, St. Louis, MO, USA. 3,3′,5,5′-tetramethylbenzidine (TMB), nicotine adenine dinucleotide phosphate (NADPH), L glutathione reduced (GSH), and oxidized (GSSG) were from SERVA Electrophoresis GmbH, Heidelberg, Germany. Hydrogen peroxide (H₂O₂), methanol, and 2,5-dihydroxybenzoic acid (DHB) were provided by Merck KGaA, Darmstadt, Germany. 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB) was purchased from Acros Organics, Geel, Belgium, while 1-methyl-2-phenylindole (MFI) and uric acid were from Alfa Aesar, Ward Hill, MA, USA. Dimethyl sulfoxide (DMSO), chloroform, and acetonitrile (C₂H₃N) were from Fisher Scientific, Waltham, MA, USA.