Fluorokine e human active mmp 1 fluorescent assay kit

The Fluorokine^® E human active MMP-1 fluorescent assay kit (R&D Systems Inc., Minneapolis, MN, USA) was used to assess MMP-1 activity according to the manufacturer’s instructions. Cells were seeded on a 60 mm culture dish at 1 × 10⁵ cells/mL. At 16 h after seeding, cells were treated with BDB (30 μM). After 1 h, the cells were exposed to UVB at a dose of 30 mJ/cm². After 24 h, culture medium was subjected to centrifugation at 1000× g for 5 min, and then 150 μL of culture supernatants was mixed with 100 μL of assay diluent buffer in 96-well enzyme-linked immunosorbent assay (ELISA) plates. The plates were shaken for 3 h at room temperature, and then unbound material was washed off. Subsequently, 200 μL of activation reagent (0.5 M APMA in DMSO) was added to each well for pro-MMP-1 activation. The plates were incubated for 2 h at 37 °C in a humidified environment. After washing, 200 μL of fluorogenic substrate was added. After another 20 h at 37 °C, fluorescence was measured using FLUOstar optima microplate reader (BMG Labtech, Cary, NC, USA) with the fluorescent cleavage product was assessed at an excitation wavelength of 320 nm and an emission wavelength of 405 nm. The MMP-1 activity was repeated three times.

MMP-1 activity was measured using a Fluorokine^® E human active MMP-1 fluorescent assay kit (R&D Systems Inc., Minneapolis, MN, USA). HaCaT cells were seeded in a 60 mm culture dish at 1.0 × 10⁵ cells/mL. After a 16 h incubation at 37 °C, the cells were treated with 20 μg/mL FFO, and 1 h thereafter with 50 μg/mL PM_2.5. MMP-1 activity was assessed according to the manufacturer’s instructions. Fluorescence was measured using a Spectra Max i3x microplate reader (Molecular Devices, San Jose, CA, USA).