96 well black assay plate

Concentrations of active MMPs were titrated by GM6001 (a highly potent broad-spectrum MMP inhibitor, EMD). In 96-well black assay plates (Corning), 60/60/250/500 nM MMP-1/-2/-7/-14cd was incubated with 0.98–1000 nM (N-)TIMPs for 30 min at room temperature, and 1 μM M-2350 substrate (Bachem) was added to start the reactions. The fluorescent signals with excitation at 328 nm and emission at 393 nm were monitored using a Synergy H4 microplate reader (BioTek). Initial velocities were calculated to determine IC₅₀, and inhibition constants (K_Is) were obtained using the following equation for slow tight-binding inhibitors [5 (link)]: $$\frac{{[I]}_t}{1-\frac{v_s}{v_0}}=K_I\frac{v_o}{v_s}+{[E]}_t$$
[I]_t is the total inhibitor concentration, [E]_t is the total enzyme concentration. v₀ is the rate of substrate hydrolysis in the absence of TIMP (M · s⁻¹), v_s is the equilibrium rate of substrate hydrolysis reached after inhibition (rate of hydrolysis in the inhibited steady state) (M · s⁻¹) for each TIMP concentration, [I]_t.

As previous described [6 (link)], intracellular ROS levels were determined with H2DCF-DA (Invitrogen, USA). Cells were seeded in 96-well black assay plate (Corning, USA), and incubated with serum-free media containing 10μmol/L H2DCF-DA for 30min. ROS levels were measured at excitation and emission wavelengths of 485 and 520 nm using a Synergy H1 Hybrid multi-mode microplate reader (Bio-Tek Instruments, USA). Control media without dye were used as background fluorescence. Cell numbers were normalized before measurement.