Human active mmp 9 fluorokine e kit

To measure the in vitro activity of released MMP-9 (Biolegend), 1 μg of MMP-9 was encapsulated in 30 μL of hydrogel, allowed to gel at the bottom of microcentrifuge tubes, and incubated in 1 mL of PBS (Gibco) (n=3). 100 μL of the supernatant was collected on day 6 and replenished with equal volume of fresh PBS. A 100-fold dilution of collected samples was used to measure MMP-9 activity using the Fluorokine® E Human Active MMP-9 kit (R&D Systems) according to manufacturer’s protocol.
For proteolytic activity of released MMP-9, MMP-9 supernatants collected from hydrogel with MMP-9 or VEGF-A+MMP-9 at day 3 release were separated on a zymography gel (Thermo Fisher). Two MMP-9 standards with concentrations of 50 and 100 ng/μL were also included. Proteolytic activity of the released MMP-9 was qualitatively evaluated as light bands on the otherwise dark zymography gel, as reported previously [29 (link)].

Endogenous MMP-9 activity was measured with a Fluorokine E Human Active MMP-9 kit (R&D Systems). For each experiment, 20 µg of protein from each sample was tested in duplicate. Fluorescence was read with a Spectramax M2 spectrophotometer (Molecular Devices, Sunnyvale, CA) at an excitation wavelength of 320 nm and an emission wavelength of 405 nm. Data were analyzed with the use of a 4-parameter regression curve (Masterplex ReaderFit; Miraibio, San Francisco, CA) and normalized to nanogram-active MMP-9 per milligram of protein in each sample.