Human active mmp 1 fluorokine e kit

Secreted active MMP-1 was measured using the Human Active MMP-1 Fluorokine^® E Kit (R&D Systems, Minneapolis, MN, USA). HaCaT ells were treated with 20 μM of DPHC for 1 h, after the 150 mJ/cm² of UVB exposure and the samples were incubated for 1 day. Culture medium was subjected to centrifugation at 1,000×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 excitation wavelength set to 320 nm and the emission wavelength set to 405 nm (Tsareva et al., 2007 (link)).