Total matrix metalloproteinase 1 (MMP-1), MMP-3, tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) or plasminogen activator inhibitor 1 (PAI-1) in cell culture medium was measured using enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems. Briefly, goat anti-human primary antibodies were coated on a 96-well plate before samples and standards were added. The sandwich ELISA was completed by adding biotinylated goat anti-human antibodies, followed by streptavidin-horseradish peroxidase and substrate solution. The optical density of each well was measured using an ELISA plate reader. Plasma LIAS levels were measured using an LIAS ELISA kit from Cusabio Biotech Co (Wuhan, China). MMP-9 in cell culture media was detected using a SensoLyte Plus 520 MMP-9 assay kit (AnaSpec, Fremont, CA, USA). Endogenously active MMP-1 activity in culture media was detected using a SensoLyte Plus MMP-1 assay kit from AnaSpec.
Sensolyte plus 520 mmp 9 assay kit
Manufactured by AnaSpec
Sourced in United States
The SensoLyte Plus 520 MMP-9 Assay Kit is a fluorescence-based kit designed to measure the activity of Matrix Metalloproteinase-9 (MMP-9) in biological samples. The kit uses a proprietary MMP-9 substrate that, when cleaved by the enzyme, releases a highly fluorescent product that can be detected.
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4 protocols using sensolyte plus 520 mmp 9 assay kit
1
Quantifying Matrix Metalloproteinases and Inhibitors
2
Quantifying MMP-9 Activity Using Fluorescent Assay
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Enzyme activity was quantified by fluorescence kit using a specific anti-MMP-9 monoclonal antibody and a fluorogenic substrate (SensoLyte Plus 520 MMP-9 Assay Kit; AnaSpec, Inc., Fremont, CA, USA), as described previously.29 (link) MMP-9 induced cleavage of the fluorogenic peptide was measured at 490 nm excitation and 520 nm emission wavelengths.
3
Determination of MMP-9 Levels in A549 Cell Conditioned Media
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Conditioned medium from A549 cells subjected to the different treatments was collected at different times (0–24 h), and MMP-9 levels were determined using NovexTM 10% zymogram Plus (gelatin) gels (Invitrogen-Life Technologies). Gels were run in a Tris-glycine SDS running buffer (Invitrogen-Life Technologies). After electrophoresis, gels were renatured, stained, and developed using the conditions described by the manufacturer. MMP-9 levels were also determined using the SensoLyte ® Plus 520 MMP-9 Assay kit (AnaSpec, Inc. Fremont, CA).
4
Quantitative Analysis of MMP-9 Activity
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The activity of the MMP-9 was assessed using the SensoLyte ® Plus 520 MMP-9 assay kit (Anaspec, Fremont, CA). Briefly, cell culture supernatants were collected after overnight serum exposure, serum starvation followed by stimulation with 100 nM PMA and 3 day invasion into organotypic matrices and centrifuged for 10 min at 1,000 x g, 4°C. A black 96-well plate covered with MMP-9 antibodies was subsequently used to capture the (pro-)MMP-9 from the sample supernatant. An activation step with APMA (2h) was performed in order to activate pro-MMP-9 and record a total signal of potential MMP-9 activity. After activation the assay substrate
(5-FAM/QXL TM 520 FRET) was added and the fluorescence reading performed using a X-Fluor Safire 2 fluorescence plate reader (λex/λem 490 nm/520 nm). The amount of MMP-9 in each sample was calculated according to the manufacturers instruction on the basis of a standard curve of the catalytic responsive enzyme. The assay was performed in 3 independent experiments for each condition.
(5-FAM/QXL TM 520 FRET) was added and the fluorescence reading performed using a X-Fluor Safire 2 fluorescence plate reader (λex/λem 490 nm/520 nm). The amount of MMP-9 in each sample was calculated according to the manufacturers instruction on the basis of a standard curve of the catalytic responsive enzyme. The assay was performed in 3 independent experiments for each condition.
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