C57BL/6 mice (wild‐type, male, 8–10 weeks of age) were purchased from the Gempharmatech (China). All experimCental procedures were approved by The Ethics Committee of University of Science and Technology of China (USTCACUC24120122042), and performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health of the United States (Eighth Edition, 2011). To establish an emphysema model, mice were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg kg−1); elastase (E7885, Sigma, USA) dissolved in PBS was intratracheally instilled (100 U kg−1).[6b ] Starting from day 7 after elastase instillation, Native EVs (2 × 109 particles per dose) and Wnt3aWG EVs (2 × 109 particles per dose) were injected via the tail vein every other day, meanwhile equal volume of PBS was injected as a control treatment. LiCl (L9650, Sigma) was administrated intraperitoneally at a concentration of 200 mg kg−1 every day.
Elastase
Manufactured by Merck Group
Sourced in United States, China, Germany, United Kingdom
Elastase is a laboratory enzyme used in various research and diagnostic applications. It is a serine protease that specifically cleaves peptide bonds on the carboxyl side of small neutral amino acids, such as alanine, valine, and serine. Elastase plays a role in the breakdown of elastin, a key component of connective tissues.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Merck Group (23 mentions)
Collagenase, by Merck Group (19 mentions)
Collagenase type 2, by Worthington (13 mentions)
Chymotrypsin, by Merck Group (12 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Collagenase type 2, by Merck Group (8 mentions)
Penicillin, by Thermo Fisher Scientific (8 mentions)
Collagenase 2, by Merck Group (7 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
Papain, by Merck Group (5 mentions)
Resveratrol, by Merck Group (5 mentions)
Penicillin, by Merck Group (4 mentions)
Streptomycin, by Merck Group (4 mentions)
Trypsin inhibitor, by Merck Group (4 mentions)
Galantamine, by Merck Group (4 mentions)
Quercetin, by Merck Group (4 mentions)
Hyaluronidase, by Merck Group (4 mentions)
Elastase, by Thermo Fisher Scientific (3 mentions)
Collagenase, by Thermo Fisher Scientific (3 mentions)
139 protocols using elastase
1
Induction and Treatment of Emphysema in Mice
2
Serine Protease Inhibition Assay
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The inhibitory activities of 8 rCvT-serpins were determined using 4 serine proteases, including trypsin (Sigma, USA), α-chymotrypsin (Sigma, USA), elastase (Sigma, USA), and subtilisin A from B. licheniformis (Sigma, USA), as previously depicted with wispy modifications (Gu et al. 2021 (link)). The residual protease activities were detected at 405 nm on a microplate reader (Thermo Fisher Scientific, USA) by the addition of 1 mM specific chromogenic substrates in 50 mM Tris-HCl buffer containing 50 mM NaCl and 5 mM CaCl2, pH 7.5, Nα-benzoyl-L-arginine 4-nitroanilide hydrochloride (Sigma B3133, USA) for trypsin, N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Sigma S7388, USA) for α-chymotrypsin, N-succinyl-Ala-Ala-Pro-Leu p-nitroanilide (Sigma S8511, USA) for elastase, and Z-Gly-Gly-Leu p-nitroanilide (Sigma C3022, USA) for subtilisin A.
3
Inhibition of Human Elastase by Elafin and Trastuzumab-Coil Elafin
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Example 54
Human elastase was purchased from Elastin Products Company, Inc. Increasing concentrations of elafin (SEQ ID NO: 258) and trastuzumab-coil elafin (CDRH3) IgG (SEQ ID NOs: 85 and 19) were incubated with elastase at room temperature, the residue activity of elastase was analyzed by the addition of fluorogenic elastase substrate MeOSuc-AAPV-AMC (EMD Millipore). The slope of the reactions were obtained by monitoring at 420 nm wavelength with 325 nm excitation on a Spectramax fluorescence plate reader. Each data point was triplicated and fit into the equation: Q=(Ki*(1+(S/Km))). Y=Vo*(1−((((Et+X+Q)−(((Et+X+Q){circumflex over ( )}2)−4*Et*X){circumflex over ( )}0.5))/(2*Et))).
4
Inhibition of Human Elastase by Elafin and Trastuzumab-Coil Elafin
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Example 54
Human elastase was purchased from Elastin Products Company, Inc. Increasing concentrations of elafin (SEQ ID NO: 258) and trastuzumab-coil elafin (CDRH3) IgG (SEQ ID NOs: 85 and 19) were incubated with elastase at room temperature, the residue activity of elastase was analyzed by the addition of fluorogenic elastase substrate MeOSuc-AAPV-AMC (EMD Millipore). The slope of the reactions were obtained by monitoring at 420 nm wavelength with 325 nm excitation on a Spectramax fluorescence plate reader. Each data point was triplicated and fit into the equation:
Q=(Ki*(1+(S/Km))).Y=Vo*(1−((((Et+X+Q)−(((Et+X+Q){circumflex over ( )}2)−4*Et*X){circumflex over ( )}0.5))/(2*Et))).
5
Isolation of Vascular Smooth Muscle Cells
6
Curcumin Regulates HDAC2 Expression
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Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium were purchased from (Thermo Fisher Scientific, Waltham, MA, USA); chromatin immunoprecipitation (ChIP) kit from Millipore (EMD Millipore, Billerica, MA, USA); anti-HDAC2 from Abcam (Cambridge, UK); anti-GAPDH from KangChen Bio-tech Inc. (Shanghai, People’s Republic of China); elastase (pancreatic from porcine pancreas) and curcumin (≥94% curcuminoid content, ≥80% curcumin) from Sigma-Aldrich Co. (St Louis, MO, USA); trichostatin A (TSA) and Cell Counting Kit-8 (CCK-8) from Beyotime Institute of Biotechnology (Haimen, People’s Republic of China); bicinchoninic acid (BCA) protein assay kit from Pierce (Rockford, IL, USA); real-time polymerase chain reaction (RT-PCR), SYBR Premix Ex Taq II (Perfect Real Time) from Takara (Tokyo, Japan); and Immobilon Western Chemiluminescent HRP Substrate from Millipore (EMD Millipore). All other chemicals were of reagent grade.
7
Isolation and Characterization of Rat Aortic Smooth Muscle Cells
8
Isolating and Transducing Mouse Smooth Muscle Cells
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SMCs were isolated from mouse bladders, from mouse intestine and from aortae by enzymatic digestion with 0.1 mg/ml elastase (Sigma) and 2 mg/ml collagenase type 2 (Worthington Biochemical Corporation) in serum free DMEM cell culture media. The SMCs were cultured in cell culture flasks containing DMEM/Ham’s F12 medium supplemented with antibiotics (penicillin and streptomycin) and 10% fetal calf serum (FCS). The flasks were placed in a water-jacketed cell incubator at 37 oC with 5% CO2 in air. SMCs were used in passages 2–5.
To examine if increased Thbs4 expression led to ATF6α activation, smooth muscle cells from mouse urinary bladder were transduced with 50 MOI Ad-CMV-m-Thbs4 or a corresponding concentration of empty vector (Ad-CMV-Null) in DMEM/Ham’s F12 media with 10% FCS and antibiotics. After 72 hours, cells were harvested for analysis. For overexpression of ATF6α we used Ad-CMV-h-ATF6α (amino acids 1–373). All viruses were obtained from Vector Biolabs. SMCs from mouse bladders were treated with 50 MOI Ad-CMV-h-ATF6α or 50 MOI Ad-CMV-Null in growth media supplemented with 2% dialyzed FCS and antibiotics for 72 hours. Before transduction, cells were starved for 2 days. Successful adenoviral transduction was confirmed using real-time quantitative PCR and western blotting. Experiments with mouse SMCs are from 3–6 independent replicates using cells from both males and females.
To examine if increased Thbs4 expression led to ATF6α activation, smooth muscle cells from mouse urinary bladder were transduced with 50 MOI Ad-CMV-m-Thbs4 or a corresponding concentration of empty vector (Ad-CMV-Null) in DMEM/Ham’s F12 media with 10% FCS and antibiotics. After 72 hours, cells were harvested for analysis. For overexpression of ATF6α we used Ad-CMV-h-ATF6α (amino acids 1–373). All viruses were obtained from Vector Biolabs. SMCs from mouse bladders were treated with 50 MOI Ad-CMV-h-ATF6α or 50 MOI Ad-CMV-Null in growth media supplemented with 2% dialyzed FCS and antibiotics for 72 hours. Before transduction, cells were starved for 2 days. Successful adenoviral transduction was confirmed using real-time quantitative PCR and western blotting. Experiments with mouse SMCs are from 3–6 independent replicates using cells from both males and females.
9
Evaluating Anti-inflammatory Agents in Cell Lines
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GSP were kindly provided by Western Animal Husbandry Co., Ltd. (Xinjiang, China). TNF-α was obtained from Peprotech (Rocky Hill, USA). Fetal Bovine Serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), trypsin and Ethylene Diamine Tetraacetic Acid (EDTA) were obtained from GIBCO (Grand Island, USA). Tissue-Tek O.C.T. Compound was obtained from Sakura Finetek Japan Co., Ltd. (Tokyo, Japan). Antibodies of Mac-2, MCP-1, mouse anti-GAPDH primary antibody and horseradish peroxidase (HRP)-conjugated secondary antibodies were all purchased from Santa Cruz (Dallas, USA). Rabbit anti-MMP-2, rabbit anti-MMP-9 and rabbit anti-elastin primary antibodies were purchased from Abcam (Massachusetts, USA). Elastase, chloral hydrate, penicillin, streptomycin, MTT and all other reagents were purchased from Sigma-Aldrich (Beijing, China).
10
Isolation and Culture of Primary VSMCs
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Primary VSMCs were isolated from WT (n = 3) and Dusp5 KO (n = 3) rats, as described previously (Fan et al., 2017 ). Briefly, the cerebral and renal vessels were isolated using the Evans blue sieving procedure (Fan et al., 2015 , 2013 ), and digested with dithiothreitol (2 mg/ml, Sigma‐Aldrich), papain (22.5 U/mL, Sigma‐Aldrich), trypsin inhibitor (10,000 U/mL, Sigma‐Aldrich), collagenase (250 U/mL, Sigma‐Aldrich), and elastase (2.4 U/mL, Sigma‐Aldrich) at 37°C. After centrifugation, the VSMCs were resuspended and seeded on autoclaved glass coverslips precoated with CellTak (Thermo Scientific) in a six‐well plate. Early passages (P2‐P3) of the primary VSMCs were used for the following experiments.
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