A modified version of an adventitially applied elastase model of AAAs was used [6 (link)]. Briefly, 9 male, C57BL/6J mice (age 14–17 weeks) underwent survival surgery under isoflurane anesthesia to expose the anterior surface of the infrarenal abdominal aorta by gentle dissection. Sterile porcine pancreatic elastase in normal saline (~8 μL at 10 U/mL, Sigma E7885) was applied onto a 1×4 mm strip of sterile gauze and placed atop the adventitial aortic surface for 10 minutes. After removing the gauze, the peritoneal space was lavaged three times with 0.5 mL of sterile normal saline. Three additional mice (age 17–21 weeks) did not undergo surgery and served as true controls since the goal of the surgery was to create a diverse set of heterogeneously evolving lesions. All surgeries and post-operative care were approved by and performed according to the policies of the Yale Institutional Animal Care and Use Committee.
E7885
Manufactured by Merck Group
Sourced in United States
The E7885 is a laboratory equipment product manufactured by Merck Group. It is a multi-purpose device designed for use in various scientific applications. The core function of the E7885 is to provide a controlled environment for conducting experiments or analyses. Further details on the specific intended use of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ibright cl1000 imaging system, by Thermo Fisher Scientific (2 mentions)
Mab050, by R&D Systems (2 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Roche (1 mentions)
Elastase, by Merck Group (1 mentions)
Dithioerythritol, by Merck Group (1 mentions)
Collagenase, by Merck Group (1 mentions)
Papain p4762, by Merck Group (1 mentions)
Maxq 4000, by Thermo Fisher Scientific (1 mentions)
Mmp 8, by Thermo Fisher Scientific (1 mentions)
Heraeus pico 17 centrifuge, by Thermo Fisher Scientific (1 mentions)
P4762, by Merck Group (1 mentions)
C8051, by Merck Group (1 mentions)
8 protocols using e7885
1
Elastase-Induced Abdominal Aortic Aneurysms
2
Elastase Modulation of Innate Immune Protein
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This experiment was carried out as per the previously reported protocol with some modifications (24 (link)). Briefly, 2μg rfhSP-D was incubated with 2.5μg porcine pancreas elastase (E7885, Sigma, USA) or 2.5 μg heat-inactivated elastase (HI elastase) for 30 minutes at 37 ° water bath. For heat inactivation, elastase was heated at 95° for 30 minutes before it was added to rfhSP-D. For inhibition of elastase activity, 5mM phenylmethylsulfonylfluoride (PMSF, Roche, Germany) was used. In addition, 2μg rfhSP-D was incubated with 2.5μg elastase in the presence or absence of 10mM CaCl2 and LPS (10μg/ml). Reactions were stopped by heating the protein sample with Laemmli buffer at 95° for 10 min. The samples were resolved on 15% SDS-PAGE under reducing conditions and the gels were Coomassie blue stained or silver stained.
3
Elastase Digestion of Recombinant LTBP4
4
Isolation of Cerebral Endothelium from Mice
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Preparation and composition of all solutions have been previously described for measurements of Vm in intact endothelium freshly isolated from mouse posterior cerebral arteries [5, 7, 21, 22 (link)]. Briefly, physiological salt solution (PSS; pH 7.4) was prepared for continuous superfusion of cerebral endothelial tubes [(in mmol/L): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 Glucose]. For dissection of brain and arteries, PSS lacking CaCl2 contained 0.1%bovine serum albumin. For isolation of intact endothelium, an enzyme cocktail [0.31 mg/mL papain (P4762, Sigma), 0.5 mg/mL dithioerythritol (D8255, Sigma), 0.75 mg/mL collagenase (C8051, Sigma), and 0.13 mg/mL elastase (E7885, Sigma)] was prepared in dissociation PSS containing reduced Ca2+ (0.1 mmol/L CaCl2) and 0.1%bovine serum albumin. The 15 mmol/L KCl solution was prepared with an equimolar decrease of NaCl accordingly in order to maintain osmolarity. All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or ThermoFisher Scientific (Pittsburgh, PA, USA) unless otherwise indicated.
5
Elastase Digestion of Recombinant LTBP4
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Recombinant human LTBP4 was incubated with anti-LTBP4 antibody for 1 hour at 4°C and then exposed to varying concentrations of elastase (U) (E7885; Sigma-Aldrich, St. Louis, MO) for 15 min at 37°C. The digestion products were separated by 4 to 15% precast polyacrylamide gel electrophoresis (4561086, Bio-Rad) and transferred to polyvinylidene fluoride membrane (1620177, Bio-Rad). Membrane was blocked in StartingBlock T20 (tris-buffered saline) Blocking Buffer for 1 hour at room temperature and then incubated overnight in His Tag primary antibody used 1:1000 (MAB050; R&D Systems, Minneapolis, MN). Secondary antibody was used at 1:2500 (115-035-003; Jackson ImmunoResearch, West Grove, PA). Pierce Pico and Femto chemiluminescent substrate was applied to membranes, and membranes were visualized using an Invitrogen iBright CL1000 Imaging System. Immunoblot bands were quantified using FIJI gel analysis tools (NIH).
6
Enzymatic Resistance of Biomaterials
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Resistance to collagenase [35 (link)] and elastase [37 (link)] degradation was also assessed. Briefly, 5 mg pieces of each material were cut and placed into Eppendorf tubes. One milliliter of Tris–HCl buffer pH 7.40 containing 50 U/mL of matrix metalloproteinase (MMP)-8 (17101015, Gibco®, Ireland) or Tris buffer pH 8.5 containing 0.1 U/mL of elastase (E7885, Sigma-Aldrich, Ireland) was added. The samples were then incubated at 37 °C under agitation in an orbital shaker (MaxQ 4000, Thermo Fisher, Ireland) at 150 rpm for 2, 4, 8, 12, and 24 h. The solubilized portion was discarded after centrifugation (Heraeus Pico 17 Centrifuge, Thermo Fisher, Ireland) at 13,000 rpm and room temperature for 10 min and the remaining pellets where weighed after overnight freeze drying (FreeZone Plus 4.5, Labconco, Thermo Fisher, Ireland). The percentage of weight loss over time was subsequently calculated for each material and enzyme.
7
Isolation and Stimulation of Mouse Aortic Smooth Muscle Cells
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Mouse abdominal aortic smooth muscle were isolated and cultured as previously described (Salmon et al. 2012 , 2013 ). Western blot analysis was performed at passage 6 to verify that SM‐actin, SM22, and SM‐MHC were expressed. For siRNA transfections, cells at passages 6–8 were plated in all six wells of a six‐well plate at 1 × 105 cells per well; 24 h later, the cells were transfected with either a single siRNA to mouse Klf4, Klf2, ZFP148, or a non‐targeting control. Cells were allowed to rest for 24 h and then were stimulated by porcine pancreatic elastase (1 unit/mL, E7885; Sigma Aldrich) or IL‐1β (10 ng/mL, 401MP; R and D Systems, Minneapolis, MN, USA) and allowed to incubate overnight at 37°C (Salmon et al. 2013 ). Cells were harvested and RNA was extracted using the TRIzol method and RT‐qPCR was performed as described previously above (Johnston et al. 2013 , 2014a ,b ; Salmon et al. 2013 ). Efficiency of siRNA transfections has been described previously (Salmon et al. 2013 , 2019 )
8
Isolation of Endothelial Tubes from Cerebral Arteries
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