Recombinant human MMP-3 (R&D Systems Inc., Minneapolis, MN, USA) was first incubated with aminophenylmercuric acetate (1 mM, 1 h, 37°C) to achieve activation. Each demineralized and pretreated dentin block was incubated with the activated MMP-3 (1 μg/ml) in a 120 μl buffer containing 100 mM Tris pH 7.2; 0.15 M NaCl; CaCl2 5 mM; 0.05% w/v Brij 35, and 0.02% w/v NaN3 (SIGMA, St Louis, MO, USA). Following 72 h incubation at 37°C, the supernatants were subjected to Western blot analysis to reveal the proteins released by MMP-3 treatment.
Mmp 3
Manufactured by Merck Group
Sourced in United States
MMP-3 is a laboratory instrument used for the detection and measurement of matrix metalloproteinase-3, an enzyme involved in the breakdown of the extracellular matrix. The core function of MMP-3 is to provide quantitative data on MMP-3 levels, which can be utilized in various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Mmp 2, by Merck Group (3 mentions)
Mmp 9, by Merck Group (3 mentions)
Mmp13, by Abcam (2 mentions)
Tgf β1, by R&D Systems (2 mentions)
Mmp 13, by Merck Group (2 mentions)
Interleukin 6 (il 6), by Merck Group (2 mentions)
Tnf α, by Merck Group (2 mentions)
Mmp 8, by Merck Group (2 mentions)
Brij 35, by Merck Group (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
Gadph, by Abcam (1 mentions)
Hif 2α, by Abcam (1 mentions)
Sox9 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti vegf a, by Bioworld Technology (1 mentions)
Carboxymethylcellulose cmc, by Merck Group (1 mentions)
Recombinant human il 1β, by Thermo Fisher Scientific (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Stat3, by Abcam (1 mentions)
13 protocols using mmp 3
1
Dentin Extracellular Matrix Proteolysis
2
Quantifying MMP-3 Activity via NIRF Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An MMP-3 digestion assay was applied to examine the in vitro reaction of the MMP-3-sensitive probe and MMP-3 enzyme through NIRF imaging. Briefly, MMP-3 (Sigma, M1677) was activated with P-aminophenylmercuric acetate (APMA) for 1 h at 37°C. After activation, an equal volume of the MMP-3-sensitive probe was mixed with the activated MMP-3/APMA solution. To prepare the solutions for the control groups, equal volumes of the MMP-3-sensitive probe was mixed with an MMP-3 solution without the activation or blank reaction buffer. All final solutions were incubated at 37°C for 30 min before being subjected to NIRF examination and quantification. For in vitro fluorescence quantification, six samples were measured in triplicate. Optical imaging of MMP-3 NIRF was performed using an optical imaging (IVIS-200 Series; Caliper, MA). Excitation and emission bandpass filters of 610-650nm and 670-700nm, respectively were employed. Both white light and NIRF images were detected using a Peltier 16-bit charge-coupled device (CCD) camera (PCO GmbH, Gottingen, Germany, −90°C cooled).
3
Chondroprotective Effects of Wogonoside
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wogonoside (purity >98 %), was purchase from Nantong Feiyu Biological Technology Co, ltd (Nantong, China). Carboxymethylcellulose (CMC) and type II collagenases were purchased from Sigma-Aldrich (St Louis, MO, USA). Recombinant human IL-1β was purchased from PeproTech (NJ, USA). The primary antibody against collagen II, aggrecan, MMP-9, MMP-13, ADAMTS5, collagen X, HIF-2α, TRAF6, p-ERK1/2, ERK1/2, p-Stat3, Stat3 and GADPH were acquired from Abcam (Cambridge, UK), MMP-3 and iNOS antibodies were obtained from Sigma-Aldrich (St Louis, MO, USA). Sox-9 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Anti-VEGF-A, Anti-Runx-2, Anti-IRAK1, goat anti-rabbit, and anti-mouse IgG-HRP was from Bioworld (OH, USA) and antibodies against COX-2, PI3K(p110), PI3K(p85), AKT, p-AKT, p-IKKα/β, IκBα, p-IκBα, p65, and p-p65 were purchased from Cell Signaling Technology (Danvers, MA, USA); Alexa Fluor®488 labeled and Alexa Fluor®594 labeled Goat Anti-Rabbit IgG (H+L) second antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA). The 4', 6-diamidino-2-phenylindole (DAPI) was obtained from Beyotime (Shanghai, China). The cell culture reagents were purchased from Gibco (Grand Island, NY, USA).
4
Quantifying Protein Secretion in Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein secretion was measured in fibroblast supernatants using commercially available sandwich ELISA kits (MMP3: Sigma-Aldrich, St. Louis, MO, USA; TGFβ1: R&D Systems, Minneapolis, MN; TIMP1: Invitrogen, Camarillo, CA, USA; TIMP4: Thermo Fisher Scientific, Waltham, MA, USA;) per manufacturer’s instructions. Supernatants were assayed undiluted except in the TIMP1 assay (diluted 1:50 with Standard Diluent Buffer).
5
Quantification of Secreted Factors in PD and TKA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A random selection of fabricated PD sheets and TKA sheets were cultured for 72 h in 3 mL of DMEM/F12 supplemented with 1% FBS and 1% AB. Supernatants were collected and centrifuged at 15,000g for 10 min to remove cell debris. The concentrations of transforming growth factor beta-1 (TGF-β1; R&D Systems), melanoma inhibitory activity (MIA; Roche), tissue inhibitor of metalloproteinases (TIMP1; R&D Systems), matrix metalloproteinase-3 (MMP3; Sigma-Aldrich), stanniocalcin-1 (STC1; Cusabio, College Park, MD, USA), and hyaluronan and proteoglycan link protein 1 (HAPLN1; US Biological, Salem, MA, USA) were measured using enzyme-linked immunosorbent assay (ELISA) kits. The signal detected for blank medium containing 1% FBS was subtracted to adjust for proteins contained in FBS. Measurements were repeated at least twice for each donor, and averages were used.
6
Quantitative Analysis of MMPs Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from cells by using a TRIzol kit; then 2 μg of RNA was used to synthesize complementary DNA (cDNA) by using reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative real-time polymerase chain reaction (qPCR) was conducted using SYBR Green (KAPA Biosystems, Woburn, MA, USA) according to the manufacturer protocol, and reactions were performed using a StepOnePlus machine (Applied Biosystems, Foster City, CA, USA). The reaction conditions were 10 min at 95°C for polymerase activation and 40 cycles of 15 s at 95°C and 60 s at 60°C. Human MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-12, MMP-13, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), purchased from Sigma-Aldrich (St. Louis, MO, USA), were used as primers to amplify target genes. The expression levels of MMPs were determined by normalizing them to that of GAPDH. The threshold cycle (Ct) was set above the nontemplate control background and within the linear phase of target gene amplification to calculate the cycle numbers at which the transcript was detected (denoted Ct). Each sample was assayed in triplicate and the data displayed represent 3 independent experiments.
7
Cytokine Stimulation of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The monocytic cell line THP‐1 and PBMCs from RA patients were cultured at 37°C for 12 hours in 96‐well plates at a density of 5 × 106 cells/ml in RPMI 1640 medium containing 10 μg/ml of matrix metalloproteinase 3 (MMP‐3; Sigma‐Aldrich), MMP‐9 (R&D Systems), ADAM‐17 (R&D Systems), and ADAMTS‐4 (R&D Systems). Soluble Sema4D concentrations were measured using an ELISA kit (MyBioSource).
8
Inflammatory Pathway Modulation: A Comprehensive Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chamazulene, recombinant rat IL-1B, CFA, primary and secondary antibodies (MMP-3, MMP-9, NF-kβ, iNOS, COX-2, TNF-α, IL-6, β-actin), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, bovine serum albumin, trypsin, streptomycin, and penicillin were purchased from Sigma Aldrich, USA. Reagents for ELISA, western blot analysis and all other CONTACT Dapeng He [email protected] chemicals were of purest analytical grade from R&D Systems (Minneapolis, USA).
9
Cytokine and MMP Quantification in Colon and Kidney
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from a 1 cm section of distal colon or kidney as previously described20 (link),37 (link)–39 (link). Concentrations of the innate cytokines IL-1α, IL-1β, IL-6, IL-10 and TNF-α (Merck Millipore, Massachusetts, USA) and matrix metalloproteases (MMP) MMP-2, MMP-3, MMP-8 and Pro-MMP9 (Merck Millipore) were determined by multiplex assay, while TIMP-1 concentrations were determined by ELISA (R&D systems, Minnesota, USA). Kidney pro-MMP-9 concentrations were determined by ELISA (R&D systems). Concentrations were normalized to total protein concentration as determined by a BCA assay (Abcam, UK) as previously described20 (link),38 (link),39 (link).
10
Antibodies and Reagents for Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse monoclonal anti-FLAG M2 (#F3165) and anti-β-actin (#A5316) antibodies were from sigma-Aldrich. Polyclonal rabbit anti-CD95 antibodies (C20, #SC-715; N18, #SC-714) were from Santa Cruz Biotechnologies. Anti-CD95 (APO1-3, #ALX-805-020) was purchased from Enzo Life Science (Villeurbane, France). Anti-Akt (#CST 9272), Anti-Akt-pS473 (D9E, #4060), anti-ERK (#9102) and anti-ERK pT202/204 (197G2, #4377) antibodies were from Cell Signaling Technology (Boston, MA). Mouse purified Anti-HA Tag (#11A3A05) was from Biolegend. Anti-CD19-PE-Cy7 (#560728), anti CD56-PE (#556647), anti CD14-PE (#555398), anti CD203c-APC (#562973), anti SIGLEC5-APC, anti-Rabbit-PE (#558416), anti-Mouse-PE (#550589), anti-FADD (#556402) and anti-CD95L (G247-4, # 556387) were acquired from BD Bioscience. Anti-Rabbit AlexaFluor555 (#A-21428) and Anti-Mouse AlexaFluor555 (#A-21422) were from Thermo Fischer Scientific. Wortmannin and UO126 were purchased from Calbiochem (Merck Chemicals Ltd., Nottingham, UK). DAPI, TMRM (Tetramethyl rhodamine methyl ester perchlorate), MTT (Thiazolyl Blue Tetrazolium Bromide), Duolink starter kit, protease and phosphate cocktails were purchased from Sigma-Aldrich. Recombinant MMP7 and MMP3 were purchased from Merck Millipore (Merck Chimie SAS, Île-de-France, France).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!