Sensolyte 520 tace activity assay kit

For the activity assay the SensoLyte 520 TACEActivity assay kit (ANASpec Inc., USA) with the fluorogenic peptide OXL^TM 520/5-FAM (ADAM17 cleavage site from TNF-α) was used. 5 × 10⁴ HEK293 cells per well were seeded in a 96-well plate. 24 hours after seeding the cells were incubated in OPTI-MEM (Thermo Fisher Scientific, UK) with the peptide substrate for 30 minutes at 37 °C and the increase of fluorescence at 538 nm (Excitation: 485 nm) was monitored as baseline activity. Afterwards cells were treated with inhibitor and/or stimulator/solvent and the activity was monitored at 37 °C for one hour. The activity of the unstimulated/stimulated cells was normalised to their baseline activity respectively.

TACE activity of the abdominal aortic tissues was measured by use of the Sensolyte 520 TACE activity assay kit (Anaspec, USA) according to the manufacturer’s instruction. Briefly, after tissues were lysed in assay buffer, incubated for 10 min at 4 °C and centrifuged at 2500 g for 10 min at 4 °C, the supernatants were collected. Equal amounts of proteins were incubated with 50μl TACE substrate for 30 min at 37 °C and changes in fluorescence were monitored by Varioskan Flash software (USA) with excitation 490 nm and emission 520 nm.

The MMP2/9 activities were evaluated using MMP2/MMP9 colorimetric Inhibitor Screening Assay kits (Abcam) under the manufacturer's instruction. In brief, 0.9 U of human recombinant MMP2 or MMP9 was mixed with 1 μM G3C12, G3C12‐NTIMP3, G3C12‐T2GNTIMP3 peptides, or 1.3 μM NNGH inhibitor as negative control. The mixture was incubated at 37°C for 60 minutes then substrate was added. The plate was read at 412 nm by infinite F50 microplate reader (TECAN). Continuous recording was made at 1 minute interval for 20 minutes. The experiment was performed in triplicate.
ADAM17 activity was determined using the SensoLyte 520 TACE Activity Assay Kit (AnaSpec). 1 μM human recombinant ADAM17 (R&D Systems) was incubated with the QXL520/ 5‐FAM FRET substrate, derived from a sequence surrounding the cleavage site of ADAM17. For inhibitor studies, 1μM TAPI‐0 or 1 μM G3C12, G3C12‐NTIMP3 and G3C12‐T2GNTIMP3 peptides were used. Active ADAM17 cleaves FRET substrate into two separate fragments resulting in an increase of 5‐FAM fluorescence which can be monitored at excitation/emission = 490 nm/520 nm measured in a fluorescence microplate reader (Beckman Coulter DTX 800) continuously every 5 minutes for 60 minutes (10).

MDSCs and NK cells were cocultured with anti-NKG2D (15 μg/mL, clone CX5, Bioxcell) antibody and ERK inhibitor FR180204 (10 μM, Beyotime) for 24 h; then, MDSCs and NK cells were isolated from the coculture system for detection of TACE activity, respectively.
TACE activity was measured using Sensolyte 520 TACE Activity Assay Kit (Anaspec, Fremont, CA, USA) at 490 nm/520 nm, following the manufacturer’s instruction.

The activity of TACE (ADAM17) was determined with the Sensolyte 520 TACE Activity Assay Kit (AnaSpec, San Jose, CA) using 20 μg of cell lysate proteins, according to the manufacturer's protocol. This assay uses a 5-FAM (fluorophore) and QXL^® 520 (quencher) FRET peptide substrate. Upon cleavage of the FRET peptide by the active enzyme, the fluorescence of 5-FAM is recovered and monitored at an excitation of 490 nm and emission of 490/520 nm. Data are expressed as the mean fluorescence intensity per microgram of total protein.

For activity assay, a SensoLyte 520 TACE activity assay kit (AnaSpec Inc., USA) with the fluorogenic peptide OXLTM 520/5-FAM (ADAM17 cleavage site from TNF-α) was used. A total of 5 × 10³ HaCat cells per well were seeded in a 96-well plate. Twenty-four hours after seeding and then incubating with ADAM17 fluorescently labelled substrate for 30 minutes at 37°C, the relative fluorescence at 520 nm (excitation: 490 nm) was monitored as baseline activity. Afterwards, the cells were treated with an inhibitor and/or stimulant and the activity was monitored at 37°C for one hour. The activity of the unstimulated/stimulated cells was normalized to their baseline activity.