For each rat, blood samples (1 ml) were taken from the left femoral vein at three time points: before MCAO surgery, before reperfusion onset, and after 5-min reperfusion. The serum was separated by centrifuging the blood samples at 3000 g for 10 min at 4 °C before measuring the targeted proteins with commercially available enzyme-linked immunosorbent assay (ELISA) kits (occludin: USCN, China; claudin-5: Biomatik, Canada; MMP-9: R&D Systems, USA): 100 μl for occludin, 50 μl for claudin-5 and 50 μl for MMP-9. The remaining serum was used for immunoprecipitation of occludin protein.
Mmp 9
Manufactured by R&D Systems
Sourced in United States, United Kingdom, Germany, China, Canada
MMP-9 (Matrix Metalloproteinase-9) is a laboratory product that functions as a recombinant human enzyme. MMP-9 is a member of the matrix metalloproteinase family and is involved in the breakdown of extracellular matrix components.
Automatically generated - may contain errors
Lab products found in correlation
Mmp 2, by R&D Systems (26 mentions)
Timp 1, by R&D Systems (19 mentions)
Interleukin 6 (il 6), by R&D Systems (11 mentions)
Tnf α, by R&D Systems (10 mentions)
Mmp 8, by R&D Systems (9 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (8 mentions)
Mmp 1, by R&D Systems (8 mentions)
Mmp 13, by R&D Systems (7 mentions)
Timp 2, by R&D Systems (7 mentions)
Mmp 3, by R&D Systems (7 mentions)
Il 1β, by R&D Systems (5 mentions)
Adam17, by R&D Systems (5 mentions)
Alginate, by Merck Group (4 mentions)
Chi3l1, by R&D Systems (4 mentions)
Il 10, by Thermo Fisher Scientific (4 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (4 mentions)
Mmp 10, by R&D Systems (4 mentions)
C reactive protein (crp), by R&D Systems (4 mentions)
Tgf β1, by R&D Systems (4 mentions)
Bms 345541, by Merck Group (3 mentions)
142 protocols using mmp 9
1
Quantifying Serum Biomarkers in Rat MCAO
2
Biomarker Analysis of Healthy Controls
3
Quantifying Serum Cortisol, DHEA, and Inflammatory Markers
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Serum was isolated using serum-separating tubes (BD Biosciences). Quantitative detection of cortisol (Alpco, Salem, NH, USA) and dehydroepiandrosterone (DHEA; Alpco), was determined by means of ELISA according to manufacturer's instructions. Levels of the primary human anti-inflammatory glucocorticoid, cortisol, are often insufficient to evaluate the function of the hypothalamus-pituitary-adrenal (HPA) axis. Therefore, we also quantified the serum concentration of DHEA, a precursor of the sex hormones testosterone and estrogen and a glucocorticoid antagonist, in order to calculate the molar ratio of cortisol to DHEA [29 (link), 30 (link)]. In addition, serum levels of IL-6, IL-10, IL-12p70, TNF-α, MMP-9, and the inflammasome effector molecule caspase-1 (R&D, Minneapolis, MN, USA) were quantified using ELISA according to manufacturer's instructions. All kits were purchased from Meso Scale Discovery (MSD, Rockville, MD, USA), unless stated otherwise. For quantitative detection of the cytokines secreted following stimulation of peripheral blood, collected plasma samples were analyzed using the following commercially available ELISA kits: IL-1β, IL-6, IL-12p70, TNF-α, IFN-α (PBL InterferonSource, Piscataway, NJ, USA), caspase-1 (R&D), and MMP-9, according to manufacturer's instructions. All kits were purchased from eBioscience, unless stated otherwise.
4
Quantification of Inflammatory Cytokines and MMP-9 in Cell Cultures
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The concentrations of TNFα, IL-1β, IL-6, IL-8, MIP1α, IL-10, and MMP-9 (R&D Systems, Minneapolis, MN, USA) present in cell culture supernatants were determined by sandwich ELISA, using human specific duo-set kits according to manufacturer’s instructions.
To coat the plates, the following capture anti-human antibodies (hAbs) were used: anti-human hAb TNFα (4 μg/mL), anti-human hAb IL-1β (4 μg/mL), anti-human hAb IL-6 (2 μg/mL), anti-human hAb IL-8 (0.5 μg/mL), anti-human hAb MIP-1α (0.4 μg/mL), anti-human hAb IL-10 (2 μg/mL), anti MMP-9 (1 μg/mL).
For the TNF-α assay, a standard curve was developed from 0.5 to 10 ng/mL with a sensitivity of 0.2 ng/mL; for the IL-1β assay, from 3.00 to 250 pg/mL; for the IL-6 assay, the curve was linear from 0.5 to 10 ng/mL with a sensitivity of 0.2 ng/mL; for IL-8, the curve was developed from 15.6 to 1000 pg/mL with sensitivity of 10 pg/mL; for MIP-1α, the curve was developed from 7.4 to 1000 pg/mL; and for IL-10, from 31.25 to 2000 pg/mL with a sensitivity of 10 pg/mL. The MMP-9 curve was performed from 31.2 to 2500 pg/mL.
To coat the plates, the following capture anti-human antibodies (hAbs) were used: anti-human hAb TNFα (4 μg/mL), anti-human hAb IL-1β (4 μg/mL), anti-human hAb IL-6 (2 μg/mL), anti-human hAb IL-8 (0.5 μg/mL), anti-human hAb MIP-1α (0.4 μg/mL), anti-human hAb IL-10 (2 μg/mL), anti MMP-9 (1 μg/mL).
For the TNF-α assay, a standard curve was developed from 0.5 to 10 ng/mL with a sensitivity of 0.2 ng/mL; for the IL-1β assay, from 3.00 to 250 pg/mL; for the IL-6 assay, the curve was linear from 0.5 to 10 ng/mL with a sensitivity of 0.2 ng/mL; for IL-8, the curve was developed from 15.6 to 1000 pg/mL with sensitivity of 10 pg/mL; for MIP-1α, the curve was developed from 7.4 to 1000 pg/mL; and for IL-10, from 31.25 to 2000 pg/mL with a sensitivity of 10 pg/mL. The MMP-9 curve was performed from 31.2 to 2500 pg/mL.
5
Quantifying Protein Biomarkers in Biological Samples
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The concentrations of MIF, HPA-1, CD138, MMP-9, IL-6 and IL-8 in the serum or cell culture medium were measured using human MIF, HPA-1, CD138, MMP-9, IL-6 and IL-8 ELISA kits (R&D Systems) following the manufacturer’s instructions. The concentrations of MIF, HPA-1, MMP-9 and CD138 in the serum or peritoneal lavage fluid of mice were measured using mouse MIF, HPA-1, MMP-9 and CD138 ELISA kits (BlueGene Biotech, Shanghai, China). NS1 ELISA was carried out using paired anti-NS1 antibodies prepared in our laboratory and was quantified by the addition of 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (R&D Systems).
6
Multiplex Cytokine Analysis of Airway Samples
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A total of 13 analytes were measured using Luminex 100 multiplex technology (Luminex Corporation, USA) in bronchoabsorption exudate and BAL samples. The measured parameters were chemokine (C-C Motif) ligand 20 (CCL20), CCL4, CCL5, chemokine (C-X-C Motif) ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, interleukin 1 beta (IL-1β), IL-6, vascular endothelial growth factor (VEGF), matrix metalloproteinase 8 (MMP-8) and MMP-9 (R&D Systems, UK).
7
Quantification of NGAL, MMP-9, and Complex
8
Calebin A and Curcumin Inhibition Study
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Calebin A was given as a kind gift by Sabinsa Corporation, East Windsor, NJ, USA. Alginate, Curcumin, BMS-345541, dithiothreitol (DTT), anti-β1-Integrin and anti-β-actin were purchased from Sigma (Munich, Germany), Epon was from Plano (Marburg, Germany). Stock solutions of DTT and BMS-345541 were prepared in PBS and further diluted in normal cell culture growth medium to obtain final concentrations. Calebin A and Curcumin were prepared in dimethylsulfoxide (DMSO) as a 5 mM stock solution and stored in small aliquots at −20 °C. During treatment, concentrations of DMSO did not exceed 0.1%. TNF-β was purchased from eBiosciences (Frankfurt, Germany) and additionally TNF-β (specific activity of 50 million U/mg) was given as a kind gift by Genetech (South San Francisco, CA, USA). Antibodies to p65-NF-κB, CXCR4, Caspase-3 and MMP-9 were from R&D Systems (Heidelberg, Germany). Ki-67 and secondary antibodies for fluorescence labelling were from Dianova (Hamburg, Germany).
9
Optimization of Biomarker Quantification Methods
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Venous blood samples were collected from each patient into a heparin sodium tube, centrifuged 1,000 rpm for 15 minutes to obtain plasma samples, and stored at −85°C until assayed. The tested parameters were measured with the enzyme-linked immunosorbent assay (VEGF, MMP-9, TIMP-1; Quantikine Human Immunoassay, R&D Systems, Inc., Minneapolis, MN, USA) and chemiluminescent microparticle immunoassay (CA 15-3; Abbott Laboratories, Abbott Park, IL, USA) according to the manufacturer’s protocols. In enzyme-linked immunosorbent assay, duplicate samples were assessed for each patient.
The intra-assay coefficient of variation of CA 15-3 is reported to be 2.2% at a mean concentration of 27.0 U/mL, standard deviation [SD] =0.6. VEGF is reported to be 4.5% at a mean concentration of 235 pg/mL, SD =10.6. MMP-9 is reported to be 1.9% at a mean concentration of 2.04 ng/mL, SD =0.039, and TIMP-1 is reported to be 3.9% at a mean concentration of 1.27 ng/mL, SD =0.05.
The inter-assay coefficient of variation of CA 15-3 is reported to be 2.6% at a mean concentration of 27.0 U/mL, SD =0.7. VEGF is reported to be 7.0% at a mean concentration of 250 pg/mL, SD =17.4. MMP-9 is reported to be 7.8% at a mean concentration of 2.35 ng/mL, SD =0.184, and TIMP-1 is reported to be 3.9% at a mean concentration of 1.28 ng/mL, SD =0.05.
The intra-assay coefficient of variation of CA 15-3 is reported to be 2.2% at a mean concentration of 27.0 U/mL, standard deviation [SD] =0.6. VEGF is reported to be 4.5% at a mean concentration of 235 pg/mL, SD =10.6. MMP-9 is reported to be 1.9% at a mean concentration of 2.04 ng/mL, SD =0.039, and TIMP-1 is reported to be 3.9% at a mean concentration of 1.27 ng/mL, SD =0.05.
The inter-assay coefficient of variation of CA 15-3 is reported to be 2.6% at a mean concentration of 27.0 U/mL, SD =0.7. VEGF is reported to be 7.0% at a mean concentration of 250 pg/mL, SD =17.4. MMP-9 is reported to be 7.8% at a mean concentration of 2.35 ng/mL, SD =0.184, and TIMP-1 is reported to be 3.9% at a mean concentration of 1.28 ng/mL, SD =0.05.
10
Western Blot Analysis of Key Signaling Proteins
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Cells lysed in NP-40 buffer and equal amounts of protein were loaded in sodium dodecyl sulfate sample buffer onto a sodium dodecyl sulfate-polyacrylamide gel, blotted onto a polyvinylidene difluoride membrane, and analyzed for expression of either MMP-9 (1:500; R&D Systems AF911), p-AKT (1:1000; Cell Signaling 4060), pan AKT (1:1000; Cell Signaling 4691), GAPDH (1:7000; Cell Signaling 2118), INSR (1:500; Cell Signaling 74118), and Hif-1α (1:250; BD Biosciences Catalog number 610959). To detect the expression of β-Actin clone AC-15 (1:2000; Catalog Number A5441) from Sigma was used. Protein sizes were determined using the Precision Plus Protein Dual Color Standards marker (Catalog Number 161-0374) from Life Technologies (Carlsbad, CA, USA). The IGF-1R Ab (0.05 µg/µl final concentration) used was MK-0646 obtained was a kind gift from the PPTC59 . The recombinant human IGF1 and IGF2 were purchased from R&D biosystems (Cat. Number 291-G1 and 292-G2) (10 ng/ml final concentration). All blots were derived from the same experiment and were processed in parallel.
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