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Mmp 1

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MMP-1 is a recombinant human Matrix Metalloproteinase-1 protein. Matrix Metalloproteinases (MMPs) are a family of enzymes involved in the breakdown and remodeling of the extracellular matrix. MMP-1 specifically cleaves interstitial collagens, such as type I, II, and III collagens.

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Lab products found in correlation

Mmp 3, by R&D Systems (8 mentions) Mmp 9, by R&D Systems (8 mentions) Mmp 13, by R&D Systems (7 mentions) Mmp 2, by R&D Systems (6 mentions) Timp 1, by R&D Systems (4 mentions) Mmp 8, by R&D Systems (4 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (3 mentions) Actinonin, by Merck Group (3 mentions) Adam17, by R&D Systems (3 mentions) Mmp 10, by R&D Systems (3 mentions) Mmp 14, by R&D Systems (3 mentions) 930 adb, by R&D Systems (3 mentions) Adam10, by R&D Systems (3 mentions) Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (2 mentions) Nobiletin, by Cayman Chemical (2 mentions) Es010, by R&D Systems (2 mentions) Nff449, by Bio-Techne (2 mentions) Vascular endothelial growth factor (vegf), by R&D Systems (2 mentions) Strataclean resin, by Agilent Technologies (2 mentions) Ab9484, by Abcam (2 mentions)

35 protocols using mmp 1

1

Dermal Fibroblast Collagen and MMP-1 Secretion

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Human primary dermal fibroblasts HDFa (ATCC, Manassas, VA, USA) were seeded in a 24-well plate at a density of 1.5 × 104 cells per well in S10 cultivation media (DMEM supplemented with 1% penicillin (100 U mL−1)–streptomycin (100 μg mL−1) (P/S) and 10% FBS). The cells were incubated for 24 h at +37 °C with 5% CO2 to allow them to attach and initiate proliferation. Fresh media containing a combination of extracts were added, and the cells were cultivated at +37 °C with 5% CO2. Media samples were collected for analysis after 24 and 72 h of incubation with the combination. The secretion of pro-collagen I and MMP-1 in the cultivation media was quantified using ELISA immunoassays employing R&D Systems DuoSet Human pro-collagen I alpha 1 and MMP-1 ELISA kits (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s recommendations. Standard dilutions of pro-collagen I (R&D Systems Minneapolis, USA) and MMP-1 (R&D Systems Minneapolis, USA) were utilized to generate the standard curve and calculate the concentrations of both analytes in the cultivation media.
Ramata-Stunda A., Boroduskis M., Pastare L., Berga M., Kienkas L., Patetko L., Skudrins G., Reihmane D, & Nakurte I. (2024). In Vitro Safety and Efficacy Evaluation of a Juniperus communis Callus Culture Extract and Matricaria recutita Processing Waste Extract Combination as a Cosmetic Ingredient. Plants, 13(2), 287.
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2

Quantification of Secreted Factors

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The supernatants were collected and analyzed for VEGF, MMP-1, MMP-13 (R&D Systems Inc., Minneapolis, MN, USA), IL-6 and IL-8 (BD, San Jose, CA, USA) using three ELISA kits (VEGF, MMP-1 and MMP-13) from R&D systems. Two kits (IL-6 and IL-8) are from BD Bioscience.
Lee Y.A., Ji H.I., Lee S.H., Hong S.J., Yang H.I., Chul Yoo M, & Kim K.S. (2014). The role of adiponectin in the production of IL-6, IL-8, VEGF and MMPs in human endothelial cells and osteoblasts: implications for arthritic joints. Experimental & Molecular Medicine, 46(1), e72-.
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3

Chondrocyte Response to MMP-1 Exposure

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Three stimulation groups were performed on DC pellets, namely the MMP-1, the CM + MMP-1, and the control group. Pellets in the MMP-1 group were cultured in chondrogenic media: DMEM-high glucose added with insulin, transferrin, and selenium (ITS-G; Thermo Fisher Scientific), 5 mg/mL linoleic acid, 10 ng/mL transforming growth factor-β (TGF-β1; R&D Systems, MN, USA), 14 mg/mL ascorbic acid , 10 -7 M dexamethasone (Sigma-Aldrich, MO, USA), 1.0 mg/mL human serum albumin (Equitech-Bio Inc., Kerrville, TX, USA), and 1% penicillin/streptomycin). Based on the concentration of MMP-1 detected in disk tissue from patients diagnosed with degenerative disk disease, concentrations of 5, 50, or 100 ng/mL MMP-1 (R&D Systems, Minneapolis, MN, USA) were selected and added to the pellet from days 14-28 during every media change. MMP-1 was added on day 14 in order to allow the cells to start producing matrix within the pellets before the matrix-degrading enzyme was added. Pellets in the CM + MMP-1 group were cultured in CM supplied with chondrogenic media in a 1: 1 ratio to replenish the nutrients depleted. MMP-1 at concentrations of 5, 50, or 100 ng/mL were added on days 14-28. Pellets treated with chondrogenic media alone without addition of MMP-1 or CM served as controls.
Hingert D., Nawilaijaroen P., Ekström K., Baranto A, & Brisby H. (2020). Human Levels of MMP-1 in Degenerated Disks Can Be Mitigated by Signaling Peptides from Mesenchymal Stem Cells. Cells, tissues, organs, 209(2-3).
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4

Nobiletin Modulates Inflammatory Signaling

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Nobiletin was purchased from Cayman Chemical (Ann Arbor, MI, USA). Recombinant human IL-1β was obtained from Peprotech (Rocky Hill, NJ, USA). Antibodies against intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were purchased from BioLegend (San Diago, CA, USA). Antibodies against phospho-p38 MAPK, phospho-extracellular signal-regulated kinase (ERK), phospho-c-Jun N-terminal kinase (JNK), phospho-IkappaB kinase (IKK)-α/β, phospho-NF-κB p65, phospho-Akt, p38 MAPK, ERK, JNK, IKK-α, NF-κB p65, Akt, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits of IL-6, IL-8, CXCL10, CCL2, CCL20, MMP-1, MMP-3, and TIMP-1 were purchased from R&D Systems (Minneapolis, MN, USA).
Hosokawa Y., Hosokawa I., Ozaki K, & Matsuo T. (2021). Nobiletin Inhibits Inflammatory Reaction in Interleukin-1β-Stimulated Human Periodontal Ligament Cells. Pharmaceutics, 13(5), 667.
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5

Cytokine and MMP Profiling in Culture

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Concentration of IL-17A, IL-17F, IL-22, IFNγ, GM-CSF, IL-6, IL-8 (all eBioscience), MMP1, and MMP3 (both R&D systems) were measured in culture supernatant using ELISA following manufacturer’s protocols.
Dankers W., den Braanker H., Paulissen S.M., van Hamburg J.P., Davelaar N., Colin E.M, & Lubberts E. (2021). The heterogeneous human memory CCR6+ T helper-17 populations differ in T-bet and cytokine expression but all activate synovial fibroblasts in an IFNγ-independent manner. Arthritis Research & Therapy, 23, 157.
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6

ELISA Analysis of Inflammatory Mediators

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Concentrations of IL-6, MMP-1, MMP-3, MMP-13 and PGE2 in medium samples were determined by enzyme-linked immunosorbent assay (ELISA) with commercial reagents (PGE2: Cayman Chemical Co., Ann Arbor, MI, USA; human IL-6: eBioscience Inc. San Diego, CA, USA; MMP-1, MMP-3, MMP-13 and mouse IL-6: R&D Systems Europe Ltd).
Nummenmaa E., Hämäläinen M., Moilanen L.J., Paukkeri E.L., Nieminen R.M., Moilanen T., Vuolteenaho K, & Moilanen E. (2016). Transient receptor potential ankyrin 1 (TRPA1) is functionally expressed in primary human osteoarthritic chondrocytes. Arthritis Research & Therapy, 18, 185.
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7

Activation and Preparation of Matrix Metalloproteinases

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Recombinant human carrier-free MMP-1, MMP-2, MMP-9, and MMP-12 (R&D Systems, Minneapolis, Minnesota) were diluted upon receipt to 75 µg/ml in 50 mM Tris, 10 mM·CaCl2, 150 mM·NaCl, 0.05% (w/v) Brij 35, and pH 7.5. MMPs were activated by incubation with 4-aminophenylmercuric acetate (APMA) at 100 mM in dimethyl sulfoxide (DMSO) (Sigma Aldrich, UK) was added to a final concentration of 1 mM. Activation incubation times at 310 K were 1 hour for MMP1-1 and MMP-2 and 24 hours for MMP-9 and MMP-12. Following incubation, enzymes were aliquoted and frozen at 193 K until required. A 5 μl aliquot was thawed and added to 600 µl of the biosensor solution, immediately prior to the NMR experiments.
Faas H.M., Krupa J.L., Taylor A.J., Zamberlan F., Philp C.J., Williams H.E., Johnson S.R., Pavlovskaya G.E., Thomas N.R, & Meersmann T. (2019). Accelerated 19F·MRI Detection of Matrix Metalloproteinase-2/-9 through Responsive Deactivation of Paramagnetic Relaxation Enhancement. Contrast Media & Molecular Imaging, 2019, 4826520.
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8

Western Blotting of Chondrocyte Pellets

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For Western blotting, chondrocytes in pellet cultures were lysed in RIPA-Benzonase buffer [22 (link)]. After the addition of the lysis buffer, the samples were left on ice for 15 minutes with vortexing every 5 minutes for 10 seconds. Lysate protein samples were then sonicated for 5 minutes, followed by centrifugation at 7000 g for 15 minutes. The protein quantity loaded on Western blot gel corresponded to 25 × 104 cells. Primary antibodies and dilutions were anti-CDKN2A/p16INK4a (ab54210; Abcam; 1:1,000) and anti-β actin (Sigma-Aldrich A228; 1:8,000). Secondary antibody used for Western blot analysis was goat anti-mouse IgG HRP conjugate (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; 115-035-003; 1:80,000). Enzyme-linked immunosorbent assays (ELISAs) were performed by using kits (IL-6, IL-8, pro-MMP13, and MMP1) from R&D Systems on supernatants stored at −20°C until analysis. Data were normalized and expressed as picograms per milliliter.
Philipot D., Guérit D., Platano D., Chuchana P., Olivotto E., Espinoza F., Dorandeu A., Pers Y.M., Piette J., Borzi R.M., Jorgensen C., Noel D, & Brondello J.M. (2014). p16INK4a and its regulator miR-24 link senescence and chondrocyte terminal differentiation-associated matrix remodeling in osteoarthritis. Arthritis Research & Therapy, 16(1), R58.
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9

Fluorogenic Protease Substrate Assay

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MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-13, MMP-14, ADAM10, ADAM17 and Mca-KPLGL-Dpa-AR-NH2 fluorogenic peptide substrates were purchased from R&D Systems (cat # 901-MP, 902-MP, 908-MP, 911-MP, 910-MP, 511-MM, 918-MP, 936-AD, 930-ADB, and ES010, respectively). All common chemicals were purchased from Sigma. NFF449 was purchased from Tocris (cat# 1391) and actinonin was from Sigma-Aldrich (cat# 01809).
Hou S., Diez J., Wang C., Becker-Pauly C., Fields G.B., Bannister T., Spicer T.P., Scampavia L.D, & Minond D. (2021). Discovery and Optimization of Selective Inhibitors of Meprin α (Part I). Pharmaceuticals, 14(3), 203.
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10

Extracellular Matrix Degradation Assay

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The MMP degradation assay was adapted from a protocol by Skjøt-Arkil et al.72 (link). The ECM-rich pellet from the CNMCS kit was solubilized in 8 M urea at pH 8 and lyophilized in 200 μg aliquots. The lyophilized ECM was resuspended in 100 mM Tris-HCl, 100 mM NaCl, 10 mM CaCl2, and 2 mM ZnOAc at pH 8.0. (Sigma-Aldrich) MMP-1, MMP-3 (901-MP, 513-MP, R&D Systems, Minneapolis, MN) MMP-2, MMP-9, MMP-13, MMP-14 (ab125181, ab168863, ab134452, ab168081, Abcam, Cambridge, MA), and MMP-7 (CC1059, Millipore) were activated according to the manufacturer’s instructions and mixed individually with 200 μg of tissue per 1 μg of either active enzyme, or MMP buffer was used as a control. Samples were mixed for 18 hours at 37°C, at which point the reaction was terminated with 25 μM of GM6001 (Millipore). Digested protein was run on a Novex 12% Tris-glycine polyacrylamide gel, stained using silver stain (Thermo) and imaged using the IN Genius Syngene Bioimaging platform (Frederick, MD).
Jansen L.E., Kim H., Hall C.L., McCarthy T.P., Lee M.J, & Peyton S.R. (2021). A poly(ethylene glycol) three-dimensional bone marrow hydrogel. Biomaterials, 280, 121270.
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