MMP9 activity was measured with the SensoLyte 520 MMP-9 Assay Kit (AnaSpec) according to the manufacturer’s instructions [19] (link), [20] . The supernatants were collected and then incubated with 4-aminophenylmercuric acetate (AMPA) and MMP9 substrate. The fluorescence intensity at Ex/Em Wave lengths of 490 nm/520 nm were used as a measure of MMP9 activity. Each experiment was repeated for three times in duplicates.
Sensolyte 520 mmp 9 assay kit
Manufactured by AnaSpec
Sourced in United States
The SensoLyte 520 MMP-9 Assay Kit is a laboratory equipment product that provides a fluorescence-based method for detecting and measuring the activity of Matrix Metalloproteinase-9 (MMP-9), an enzyme involved in various biological processes. The kit includes the necessary reagents and components to perform the assay.
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9 protocols using sensolyte 520 mmp 9 assay kit
1
Quantifying MMP-9 Activity Assay
2
Quantifying MMP-9 Activity Assay
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MMP9 activity in cell culture media was determined per manufacturer’s instructions using an activity assay kit with a fluorescent substrate cleaved by MMP-9 (SensoLyte 520 MMP9 Assay Kit, AnaSpec, Inc., Fremont, CA, USA). GM1489 (MMP1 inhibitor; EMD Millipore, Billerica, MA, USA) was supplemented to the reaction mixture at 0.1–10 μM to suppress nonspecific degradation of the substrate by MMP-1.
3
Quantifying MMP9 Activity in Cells
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The activity of MMP9 was measured using the SensoLyte® 520 MMP9 assay kit (AnaSpec, Fremont, CA, USA) according to the manufacturer's protocol. The cells were seeded in 6-well plates at a density of 3×105 cells/well and treated with FZKA (0, 1, 2 and 3 mg/ml) for 24 h. Subsequently, the cell culture media supernatant was collected and centrifuged at 1,000 × g for 15 min at 4°C. The MMP containing samples were incubated with APMA (component C) at a final concentration of 1 mM in the assay buffer (Component D) and were incubated for 2 h at 37°C in order to activate pro-MMPs. The working solutions were then prepared by diluting the MMP9 substrate 1:100 in assay buffer. The reagents: 50 µl MMP9 containing sample and 50 µl MMP9 substrate solution, were mixed in a 96-well plate by gentle agitation for 30 sec. The reactions were incubated at 37°C for 1 h and fluorescence intensity was measured at excitation/emission=490/520 nm. The experiment was repeated three times.
4
Measuring MMP-9 Activity Using Fluorescent Assay
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MMP‐9 activity was measured using the SensoLyte 520 MMP‐9 Assay Kit according to the manufacturer's protocol (AnaSpec) as previous report 10 . Briefly, tissues were homogenized with the assay buffer containing 0.1% Triton X‐100, and centrifuged for 15 min. at 10,000 g at 4°C. The supernatant was collected. And the supernatants were incubated with 4‐aminophenylmercuric acetate for 1 hr at 37°C. Then, MMP‐containing samples were added to a 96‐well plate (50 μl/well), and MMP‐9 substrate (50 μl/well) was added to the sample and control wells. After shaking the plate gently for 30 sec., the reagents were incubated at 37°C for 1 hr. Then, 50 μl of stop solution was added and mixed, and the fluorescence intensity was measured at an excitation wavelength of 490 nm and an emission wavelength of 520 nm.
5
MMP-9 Enzymatic Activity Assay
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The SensoLyte 520 MMP-9 Assay Kit (Ana Spec, Inc., #71155, Fremont, CA) was used to determine the enzymatic activity of MMPs with and without oligonucleotides. Purified MMP protein was added at the indicated concentrations in MMP Dilution Buffer (50 mM Tris–HCl, 200 mM NaCl, 5 mM CaCl2, 1 µM ZnCl2, 0.05% Brij-35, and 0.05% NaAzide, pH 7.0) with the addition of the indicated concentrations of oligonucleotides. Hundred microliters of hMMP9/oligonucleotide samples were supplemented with 1 µM ZnCl2 and 2 mM APMA, mixed, added to a black 96-well plate, and incubated at 37°C for 1 h. APMA was omitted from the reaction when assaying for the catalytic domain — MMP9 since preactivation was not required. After incubation, a final concentration of 20 µM of the fluorogenic substrate (Enzo Life Sciences) in 100 µl of 2× assay buffer (100 mM HEPES, 20 mM CaCl2, and 0.1% Brij-35, pH 7.0) was added to the activated MMP protein and reaction kinetics were quantified using a Synergy 2 microplate reader (BioTek, Inc., Winooski, VT) with an excitation wavelength/bandwidth of 360/40 and an emission wavelength/bandwidth of 460/40 at 37°C for 1 ½ h. Unless otherwise indicated, the final concentration of MMP9 protein was 2 nM with variable concentrations of ssDNA in the final assay reaction.
6
MMP-9 Activity Assay of Glycosylation Mutants
7
Quantifying MMP-9 Activity in T24 Cells
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The T24 cells with different treatments (5 × 105/well) were seeded onto 12-well plates and filled with 500 μl medium for 48 hr. The supernatants were collected after centrifugation at 2500 rpm (4°C) for 10 min. MMP-9 activity was assessed by SensoLyte® 520 MMP-9 assay kit (ANASPEC, San Jose, CA, USA) according to the manufacturer’s instructions. The MMP-9 activity was monitored at excitation/emission = 490 nm/520 nm by fluorescence spectrometer (Thermo Scientific, Waltham, MA, USA).
8
Quantifying Tumor MMP-9 Activity
9
Quantifying MMP-9 Activity in Stimulated Brain Extracts
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