Elastase type 3

Manufactured by Merck Group

Sourced in United States

Elastase type III is a laboratory enzyme used in biochemical research and analysis. It is a serine protease that specifically cleaves peptide bonds on the carboxyl side of small neutral amino acids. This enzyme is commonly used for the digestion and processing of protein samples in various experimental procedures.

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Lab products found in correlation

Bovine serum albumin (bsa), by Merck Group (2 mentions) Collagenase type 2, by Worthington (2 mentions) Collagenase type 2, by Merck Group (2 mentions) Collagenase type 1, by Worthington (2 mentions) Desmin, by Agilent Technologies (1 mentions) Collagenase cls1, by Worthington (1 mentions) Alexa fluor 594 goat anti mouse, by Thermo Fisher Scientific (1 mentions) Dmem culture medium, by Thermo Fisher Scientific (1 mentions) Sm α actin, by Merck Group (1 mentions) Alexa fluor 594 goat anti rabbit, by Thermo Fisher Scientific (1 mentions) Sm22α, by Abcam (1 mentions) Collagenase cls 2, by Worthington (1 mentions) Soybean trypsin inhibitor, by Worthington (1 mentions) Dmem f12, by Thermo Fisher Scientific (1 mentions) Collagenase type 1, by Merck Group (1 mentions) Il 1β, by BioLegend (1 mentions) Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions) Ambion silencer select validated sirna, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Elastase (139 citations) Type III (4 citations)

6 protocols using elastase type 3

Isolation and Characterization of Aortic Vascular Cells

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6-10 week old wild type, OPG−/−, ApoE−/−OPG−/− and ApoE−/−OPG+/+ aortas were incubated in an enzyme mix solution containing 2 mg/ml BSA (Sigma), 1 mg/ml collagenase CLS-1 (Worthington), 0.375 mg/ml soybean trypsin inhibitor (Worthington), 0.125 mg/ml elastase type III (Sigma) for 5 min at 37°C. The adventitia was then removed, the endothelium stripped and the media cut into small pieces that were dispersed in a mixture of 0.6 mg/ml collagenase CLS-2 (Worthington) and 0.25 mg/ml elastase type III (Sigma) in culture medium containing FBS and incubated at 37°C for 1h. The cell suspension was centrifuged and re-suspended in DMEM culture medium (Invitrogen) containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 20% FBS. The cells were split when confluent and cultured in growth media with 10% FBS after passage 3. The cells used for the experiments were primary cultures and subcultures of 3-9 passages.
Cells at passage 1 were plated on glass chamber slides (Lab-Tek, Rockford, IL) and used for cell characterization by immunofluorescence. Cells were stained with antibodies against SMα-actin (Sigma), SM22α (Abcam), desmin (Dako, Carpinteria, CA) and CD31 (BD Bioscience, San Jose, CA). Alexa fluor 594 goat anti-mouse, alexa fluor 594 goat anti-rabbit, and alexa fluor 594 rabbit anti-goat (Invitrogen) were used as secondary antibodies.

Callegari A., Coons M.L., Ricks J.L., Rosenfeld M.E, & Scatena M. (2014). Increased Calcification in Osteoprotegerin Deficient Smooth Muscle Cells: Dependence on Receptor Activator of NF-κB Ligand and Interleukin-6. Journal of vascular research, 51(2), 118-131.

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Isolation and Culture of Aortic VSMCs

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The vascular smooth muscle cells (VSMCs) of the descending thoracic aorta were prepared from the 8-week-old control and defeated mice as previously described [25 (link)] because the amount of tissue in the infrarenal aorta was much smaller than the amount in the thoracic aorta. The media was excised from the descending thoracic aorta, followed by incubation with 1 mg/mL collagenase type II (Worthington Biochemical Corporation. Lakewood, NJ, USA) to remove endothelial and adventitial cells. The aortic medias were spread in medium containing 1 mg/mL collagenase type II, 0.5 mg/mL elastase type III (Sigma-Aldrich, St. Louis, MO, USA), and 20% fetal bovine serum (FBS). Cell suspensions were centrifuged at 500× g for 5 min, and the cell pellets were resuspended in Dulbecco’s modified eagle medium (DMEM) containing 100 U/mL penicillin, 100 μg/mL streptomycin, and 20% FBS. VSMCs were stimulated with 20 ng/mL PDGF-BB (PMG0045; Thermo Fisher Scientific) or 10 ng/mL TGF-β (766-MB; R&D Systems, Minneapolis, MN, USA), as shown in each experiment.

Kubota H., Yamada H., Sugimoto T., Wada N., Motoyama S., Saburi M., Miyawaki D., Wakana N., Kami D., Ogata T., Ibi M, & Matoba S. (2022). Repeated Social Defeat Enhances CaCl2-Induced Abdominal Aortic Aneurysm Expansion by Inhibiting the Early Fibrotic Response via the MAPK-MKP-1 Pathway. Cells, 11(4), 732.

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Isolation of Mouse Aortic VSMCs

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Mouse VSMCs were isolated from the aorta of 4-6 week old WT and sam68^−/− mice as described [24 (link)]. Thoracic aortas were harvested and after removing surrounding connective tissues, cut into small pieces and digested in collagenase type I (1 mg/ml, Worthington, #LS004214) and Elastase type III (0.125mg/ml, Sigma Aldrich, #E0127) at 37°C for 40 min, pipetting vigorously every 10 min. Then cell suspension was spun down at 1400 rpm for 5 min at 4°C, re-suspended in 2 mL DMEM/F12 (Thermo Fisher Scientific,#11320082) with 10%FBS and 1% Penicillin/Streptomycin, and seeded in 6 well plates for culture in the incubator. Passages 4 to 7 VSMCs at 70-80% confluence was used for experiments.

Han S., Xu S., Zhou J., Qiao A., Boriboun C., Ma W., Li H., Biyashev D., Yang L., Zhang E., Liu Q., Jiang S., Zhao T.C., Krishnamurthy P., Zhang C., Richard S., Qiu H., Zhang J, & Qin G. (2019). Sam68 impedes the recovery of arterial injury by augmenting inflammatory response. Journal of molecular and cellular cardiology, 137, 82-92.

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Isolation of Primary Detrusor Smooth Muscle Cells

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Primary detrusor smooth muscle cells from each model group were separated as previously described [14 (link)]. Briefly, isolated bladder tissue from the sacrificed rats were cut to small pieces, the muscle layer were cleared from the mucosa, epithelium, and other attached tissue under a dissection microscope. Separated muscle tissue were cut into fine sand like particles and digested with 10 U/ml elastase (type III), 150 U/ml collagenase (type I), and 2.0 mg/ml bovine serum albumin (all purchased from Sigma). After incubation at 37°C for 90 min (mix every 5 min), the cells were filtered through a 100 μM cell strainer and centrifuged at 3000 rpm for 5 min. The obtained smooth muscle cells were resuspended in DMEM medium supplemented with 10% fetal bovine serum (Hyclone) and seeded into indicated dish. Once the cells adhere to the wall, they were harvested, measured, or immune-stained.

Zhu X., Tao F., Zhang L., Yu Y., Xu Y., Yang G., Wei Z., Cheng Y., Yin X., Zhang X., Wei W, & Wang A. (2022). Astragaloside IV Protects Detrusor from Partial Bladder Outlet Obstruction-Induced Oxidative Stress by Activating Mitophagy through AMPK-ULK1 Pathway. Oxidative Medicine and Cellular Longevity, 2022, 5757367.

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Aortic VSMC Isolation and Calcification Induction

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Aortic VSMCs were prepared from 8-week-old O-ND and O-HFD as described previously [25 (link)]. The media was stripped from the thoracic aorta under a dissection microscope, followed by incubation with 1 mg/mL collagenase type II (Worthington Biochemical Corporation. Lakewood, NJn USA) to remove residual endothelial and adventitial cells. Aortic media were then dispersed in a medium containing 1 mg/mL collagenase type II, 0.5 mg/mL elastase type III (Sigma-Aldrich), and 20% fetal bovine serum (FBS). Cell suspensions were centrifuged at 500 g for 5 min, and the cell pellets were resuspended in DMEM containing 100 U/mL penicillin, 100 Ag/mL streptomycin, and 20% FBS. Aortic VSMCs were seeded at a density of 1 × 10⁵ cells/mL for primary culture and split at 1:2 at confluency. The cells used for the experiments were obtained from the primary culture to passage 5. Subcultured SMCs were maintained in DMEM containing 1% penicillin/streptomycin and 20% FBS. To induce transformation, cells were cultured in DMEM containing 3% FBS and inorganic phosphate (Pi), as indicated below. VSMC calcification was induced by treatment with calcification media supplemented with NaH₂PO₄/Na₂HPO₄ to a final concentration of 2.6 mmol/L phosphate with 3% FBS, with or without IL-1β (BioLegend) for 14 d.

Miyawaki D., Yamada H., Saburi M., Wada N., Motoyama S., Sugimoto T., Kubota H., Wakana N., Kami D., Ogata T, & Matoba S. (2022). Maternal high-fat diet promotes calcified atherosclerotic plaque formation in adult offspring by enhancing transformation of VSMCs to osteochondrocytic-like phenotype. Heliyon, 8(9), e10644.

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Isolation and Culture of Rat Aortic SMCs

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Rat aortic SMCs were isolated and cultured as described previously.^{20 (link)} Briefly, rat aortas were excised and dissected free of adventitia and fat. The vessels were further digested by collagenase type I (Worthington Biochemical, 1 mg/ml) and elastase type III (sigma 0.125 mg/ml) at 37°C in a CO₂ humidified chamber. Then, cell suspensions were centrifuged and the cell pellets were re suspended and grown in DMEM with 10% fetal bovine serum and penicillin and streptomycin (1%) in 5% CO₂ at 37°C. The SMC phenotype was determined as described above. Cells of passages 2–3 were used for the short interfering (si)RNA transfection study. SMCs were transfected with control siRNA or siRNA targeting rat calpain-2 or filamin A (Ambion - Silencer Select validated siRNA, ThermoFisher Scientific) sequences using RNAiMax lipofectamine transfection reagent (ThermoFisher Scientific). After 48 h of transfection, cells were treated with either vehicle or AngII (100 nM) for 24 h and then harvested for western blot analyses.

Muniappan L., Okuyama M., Javidan A., Thiagarajan D., Jiang W., Moorleghen J.J., Yang L., Balakrishnan A., Howatt D.A., Uchida H.A., Saido T.C, & Subramanian V. (2021). Inducible Depletion of Calpain-2 Mitigates Abdominal Aortic Aneurysm in Mice. Arteriosclerosis, thrombosis, and vascular biology, 41(5), 1694-1709.

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