Recombinant mmp 9

Human embryonic kidney cell line HEK293 and human lung carcinoma cell line A549 were purchased from American Type Culture Collection (USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% (v/v) heat-inactivated bovine calf serum (BCS; Thermo Fisher Scientific, Waltham, MA, USA) and 100 units/mL penicillin and streptomycin (Invitrogen, Calsbad, CA) at 37 °C in a humidified atmosphere of 5% (v/v) CO₂. The Expi293F cells (Thermo Fisher Scientific, Waltham, MA, USA) were cultured in Expi293 Expression Medium at 37 °C in a humidified atmosphere of 8% CO₂. 2,2′-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS) and 30% hydrogen peroxide were from Sigma-Aldrich. Horseradish peroxidase (HRP)-conjugated goat anti-human or -mouse IgG Fc and HRP-conjugated goat anti-mouse IgG F(ab′)₂ secondary antibodies were from Jackson Immunoresearch Laboratories (West Grove, PA). Recombinant Interleukin (IL)-1β, IL-6 and human IL-6 DuoSet ELISA kit were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant MMP-9 and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

HEK 293 cells stably expressing PAR1-SEAP (kindly provided by Dr. Mosnier, The Scripps Research Institute, La Jolla, CA, USA) were used for reporter assays as described previously [35 (link)]. PAR1-SEAP cells were incubated with 100 nM recombinant MMP9 (pre-activated, Sigma, St. Louis, MO, USA), 0.1 U/ml thrombin, or solvent control for 30 min in serum-free Opti-MEM (Gibco, Thermo Fischer Scientific, Waltham, MA, USA). Next, the supernatant was removed, and alkaline phosphatase activity was measured according to the manufacturer’s instructions using a Synergy HT Biotek Microplate Reader (Biotek Instruments, Winooski, VT, USA).