MMP1 activity in the cell culture medium was measured using a SensoLyte Plus 520 MMP1 assay kit (Anaspec, Fremont, CA) according to the manufacturer's protocol. The cell culture medium of mesothelial cells treated with EVs was collected 48 h after changing the medium. The CM was used after centrifugation at 2,000 g for 10 min at 4 °C. The medium was incubated with a substrate that fluoresces when cleaved by MMP1. The fluorescence was measured by a microplate reader (Gen5, BioTek Instruments). The concentration of active MMP1 in the medium was determined using a calibration curve.
Sensolyte plus 520 mmp 1 assay kit
Manufactured by AnaSpec
Sourced in United States
The SensoLyte Plus 520 MMP-1 Assay Kit is a fluorescence-based kit designed to detect and quantify the activity of Matrix Metalloproteinase-1 (MMP-1) in biological samples. The kit utilizes a proprietary fluorogenic peptide substrate that is cleaved by MMP-1, resulting in the release of a fluorophore, which can be measured using a fluorescence reader.
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4 protocols using sensolyte plus 520 mmp 1 assay kit
1
Quantifying MMP1 Activity in Cell Culture
2
Quantification of Pro-MMP1, PGE2, and IL1RN
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Pro-MMP1 was detected with the SensoLyte Plus 520 MMP-1 Assay Kit (AnaSpec, Freemont, CA, USA). PGE2 and IL1RN levels were measured with the Biotrend PGE2 Enzyme Immunoassay Kit (Biotrend Chemikalien, Köln, Germany) and the Human IL-1ra/IL-1F3 Quantikine ELISA Kit (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions. Fluorescence and absorbance values were measured on a Tecan Infinite M200 reader (Tecan, Crailsheim, Germany).
3
Quantifying Human MMP1 Activity
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To determine the activity of human MMP1, cell culture supernatants were collected after 48 h of culture. The activity of human MMP1 were measured by a SensoLyte® Plus 520 MMP-1 Assay Kit (AnaSpec, San Jose, CA, USA; AS-72012) according to the manufacturer’s instructions. A specific anti-MMP-1 monoclonal antibody was used in combination with an MMP1 fluorogenic substrate, 5-FAM/QXL®520 FRET peptide. The fluorescence signal is monitored at Ex/Em = 490 nm/520 nm upon MMP-1-induced cleavage of the FRET substrate.
4
Quantifying MMP1 Activity in Conditioned Media
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The MMP1 activity in conditioned media of control, IL-1α (1 ng/ml), and a combination of IL-1α and TGFβ (2 ng/ml) was determined using the SensoLyte Plus 520 MMP-1 Assay Kit (AnaSpec, San Jose, CA). Briefly, the conditioned media were collected from PC013, BxPC-3, and CAPAN2 cells after 3 days of culture in DMEM supplemented with 1% FBS. One hundred microliters of conditioned media was added to MMP1 antibody precoated microplate wells. The MMP1 substrate 5-FAM/QXL520 fluorescence resonance energy transfer (FRET) peptide was added to the wells, and after 16 hours of incubation, the fluorescence intensity representing the MMP1 activity was measured at 490/520-nm wavelength using EnVision Multilabel Reader (Perkin Elmer, Waltham, MA).
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