Cells and media were collected in falcon tubes and centrifuged. Supernatant was transferred to a new falcon tube and cells were washed with PBS and centrifuged. Equivalent amounts of total protein from cells or media (100 μg) per cell line were used to measure MMP-9 levels. MMP-9 expression was performed using a commercial kit (Human MMP-9 ELISA Kit, BMS2016–2, ThermoFisher, Waltham, MA) according to the manufacturer’s instructions. MMP-9 expression is presented as a mean ± SD of two independent experiments. Expression in cells. Cells were lysed (4 °C, 1h) with RIPA buffer (50 mM Tris-Cl, 150 mM NaCl, 1% NP-40 (Igepal CA-630, Sigma-Aldrich, St. Louis, MO), 0.5% Sodium Deoxycholate, 0.1% SDS, 1 mM DTT) containing a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Lysates were centrifuged (14,000 × g, 5 min) and total protein concentration was measured by Bradford assay. Expression in Media. Media was filtrated through 50K cut-off filters (Amicon® Ultra-15 Centrifugal Filter Units, Millipore Sigma, Burlington, MA) according to the manufacturer’s instructions. Only proteins with molecular weights higher than 50 KDa were used for MMP-9 expression assays.
Human mmp 9 elisa kit
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
The Human MMP-9 ELISA Kit is a laboratory assay used for the quantitative measurement of matrix metalloproteinase-9 (MMP-9) levels in human biological samples. The kit utilizes the enzyme-linked immunosorbent assay (ELISA) technique to detect and quantify MMP-9 concentrations.
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Lab products found in correlation
Igepal ca 630, by Merck Group (2 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (2 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Human igf1 simplestep elisa kit, by Abcam (1 mentions)
Human vegf simplestep elisa kit, by Abcam (1 mentions)
Human mmp 2 elisa kit, by Thermo Fisher Scientific (1 mentions)
Il 1 beta human uncoated elisa kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Tnf alpha human uncoated elisa kit, by Thermo Fisher Scientific (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
8 protocols using human mmp 9 elisa kit
1
Quantification of MMP-9 Expression in Cells and Media
2
Quantification of MMP-9 Expression
3
Quantification of Human MMP-9 by ELISA
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The test was performed with the Human MMP-9 ELISA kit from Invitrogen, BenderMedSystems GmbH, Vienna, Austria, as described earlier [52 (link)]. Briefly, the 96-well assay plate coated with anti-MMP-9 antibody was washed and then loaded with 100 µL standards (9 serial concentrations in the range of 0.017–7500.00 ng/mL, in duplicates), 100 µL of assay buffer as blank, 100 µL of samples, in duplicates. A total of 50 µL biotin-conjugated anti-MMP-9 antibody solution was dispensed in each well, and the plate was incubated with shaking for 2 h. The plate was washed four times, 100 µL of Streptavidin-HRP solution was added to each well, and the plate was incubated for 1 h. After another wash cycle, the wells were loaded with 100 µL TMB substrate and after 15 min, a stop solution was added to the plate, and the samples were read with the ELISA reader, at 540/450 nm. The standard curve was created, and individual concentrations were plotted by the Magellan software of the ELISA Equipment.
4
Quantification of MMP-9, VEGF, and IGF1
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Measurement of MMP-9, VEGF, and IGF1 protein content was conducted using a Human IGF1 SimpleStep ELISA® Kit (Abcam, Cambridge, UK), Human VEGF SimpleStep ELISA® Kit (Abcam, Cambridge, UK), and Human MMP-9 ELISA Kit (Invitrogen, Waltham, MA, USA), respectively. All procedures were conducted in accordance with the manufacturer’s protocol. The endpoint reading (color intensity) of all analytes was measured at 450 nm and protein concentration was calculated from the standard curve.
5
Quantifying MMP-2 and MMP-9 Levels
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The concentrations of MMP-2 and MMP-9 were further evaluated in both cell lines. Cell culture supernatants were collected at 48 h after transfection. MMP-2 and MMP-9 concentrations were measured using human MMP-2 ELISA Kit and human MMP-9 ELISA Kit (Invitrogen Corporation, Carlsbad, United States) according to the manufacturer’s instructions. The optical density (OD) value at 450 nm was record using a microplate reader.
6
Quantification of MMP-9 and DENV2-NS1
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The concentration of culture supernatants and MMP-9 or NS1 were measured by Human MMP-9 ELISA Kit (Invitrogen) or DENV2-NS1 ELISA Kit (Arigo biolaboratories) according to manufacturer’s instructions.
7
Quantitative MMP-9 Expression Analysis
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The expression of MMP-9 was analyzed using commercially available quantitative test ELISA (Thermo Fisher Scientific, MMP-9 Human ELISA Kit, BMS2016-2) following the manufacturer’s instructions.
8
Protein Extraction and Quantification
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Constructs were isolated and homogenized in M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher) containing 1X protease inhibitor cocktail (Sigma) for 30 minutes on ice to ensure complete cell lysis. Cell lysates were centrifuged at 12,000 rpm for 15 minutes at 4 °C to pellet insoluble debris, and supernatants were aliquoted and stored at −20 °C until further use. Protein expression was measured using commercial ELISA kits: IL-1 beta Human Uncoated ELISA Kit (Thermo Fisher, 88-7261-22), TNF alpha Human Uncoated ELISA Kit (Thermo Fisher, 88-7346-22), and MMP9 Human ELISA Kit (BMS2016-2) according to the manufacturer’s protocol.
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