All experiments involving mouse tissues complied with the ethical regulations of, and were approved by, the Animal Ethics Committees of the Garvan Institute of Medical Research/St Vincent’s Hospital and Sydney Local Health District. Arterial tissue was obtained from 8- to 12-week-old male C57BL/6J (Animal Resources Centre) and PKG1α C42S knock-in mice (C57BL/6N background)9 (link). Animals were housed in rooms with a 12 h light/dark cycle corresponding to sunrise and sunset of approximately 6 am and 6 pm, respectively. The room temperature was maintained between 20 and 26 °C with humidity set between 40 and 70%. Where indicated, mice were subjected to endotoxemia by intraperitoneal injection of 7.5 mg kg−1 body weight LPS (0111:B4, Sigma-Aldrich L4130) for 16 h. Mice were anesthetized using isoflurane and tissue collected after cardiac puncture, exsanguination, and perfusion using sterile PBS in the absence and presence of NEM as indicated. Once collected, arterial tissue was placed into ice-cold Krebs-Henseleit buffer (Sigma-Aldrich K3753, supplemented with 2.5 mM CaCl2, 10 mM EDTA, 2.1 mg mL−1 sodium bicarbonate and adjusted to pH 7.4) and dissected free of adipose and connective tissue. Stomachs were also collected and immediately snap frozen for further analysis.
K3753
Manufactured by Merck Group
Sourced in United States, Sao Tome and Principe, Japan
K3753 is a lab equipment product from Merck Group. It is a precision instrument designed for use in scientific laboratories. The core function of K3753 is to perform accurate measurements and analysis of various samples. Further details on the intended use or specific applications of this product are not available.
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11 protocols using k3753
1
Arterial Tissue Isolation and Endotoxemia Model
2
Isolation of Piglet Colonic Epithelial Cells
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Colon samples were obtained from slaughterhouse material of approximately 10-week-old piglets. A 20 cm section of mid-sigmoid colon was obtained immediately after slaughter, and intestines were placed in aerated Krebs Henseleit Buffer containing 5 mM glucose (hereafter referred to as modified-KHB, K3753, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 2.5 g/L Bovine serum albumin (BSA, A7906, Sigma-Aldrich, St. Louis, MO, USA). After this, the intestines were flushed with modified-KHB. Then, they were inverted, and a sac was created using dialysis clamps by filling them with modified-KHB. The sacs were incubated for 20 min in Ca2+-free KHB buffer containing 20 mM EDTA and 10 mM DTT in a shaking 37 °C water bath. Following this washing step, intestines were re-verted and filled with an isolation buffer containing Ca2+-free KHB buffer, 2.5 g/L BSA and 400 U/mL hyaluronidase type IV (3884, Sigma-Aldrich, St. Louis, MO, USA). After a fifteen-minute incubation, the intestines were gently massaged and cells were collected, washed and counted using a Cellometer K4 (Nexcelom Bioscience, Lawrence, MA, USA), and viability was simultaneously assessed by staining with ViaStain (CS2-0106, Nexcelom Bioscience, Lawrence, MA, USA). Cells were taken up in KHB medium containing 1 mM HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid) and 2.5 mM glucose, pH 7.4.
3
Evaluating Liver Damage in Perfused Rat Model
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The isolated perfused rat liver (IPRL) system was used to assess liver damage and their functional integrity according to the standardized conditions.28 , 29 Briefly, oxygenated reperfusion was performed for 120 minutes via the PV at a constant flow of 3 mL/g liver/minute with 150 mL of oxygenated Krebs‐Henseleit bicarbonate (KHB; K3753, Sigma‐Aldrich Inc., St. Louis, MO) at 37°C. Carbogen (95% O2 + 5% CO2) was used for oxygenation, and the prehepatic partial pressure of oxygen was continuously maintained above 500 mm Hg. hyaluronic acid (CAS.9067‐32‐7; Wako Pure Chemical Industries, Ltd., Japan) was supplemented into the KHB (1 mg/L), and the perfusate was taken to evaluate transaminase release, oxygen consumption, and hyaluronic acid clearance. Total bile production and portal vein pressure (PVP) were measured throughout reperfusion.28
4
Cardiac Muscle Preparation for Contractility
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All animal procedures were in accordance with Schedule 1 of the UK Animal (Scientific Procedures) Act 1986. Adult Wistar rats (200–250 g) were sacrificed by cervical dislocation. The hearts were excised and rinsed free of blood with Krebs-Henseleit solution (K3753, Sigma, St. Louis, MO) oxygenated with a carbogen mixture of 95% O2% and 5% CO2. Suitable trabeculae (free running, unbranched, diameter <250 µm) were dissected from the right ventricle in Krebs-Henseleit solution containing 25 mM 2,3-butanedione-monoxime, permeabilised in relaxing solution (see below) containing 1% Triton X-100 for 30 min, and stored in relaxing solution for experiments.
5
Vascular Reactivity Assessment in Mouse Aorta
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Pressurised wire myography was used to evaluate the vascular reactivity on mouse aortic ring segments as described previously with minor modifications [43 (link)]. In brief, experiments were performed in Krebs’ buffer (Sigma-Aldrich, K3753) supplemented with 3.4 μM CaCl2 and diffused with 95% O2, 5% CO2 at 37 °C. After mounting and stabilisation, 2 mm vessel segments were constricted with 1 × 10−6 M norepinephrine (NE, Sigma-Aldrich, 74480) and then assessment of relaxation potential in response to increasing doses of acetylcholine (Ach, Sigma-Aldrich, A6625) or sodium nitroprusside (SNP, Sigma-Aldrich, PHR1423). Experiments were performed on n ≥ 4 aortas in duplicate, from each of the respective feeding cohorts.
6
Ex Vivo Rat Vascular Relaxation
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To perform the ex vivo relaxation experiments and the immunohistochemical staining of rat blood vessels, the rats were sacrificed by bleeding from the carotid arteries under isoflurane anesthesia. Aortic rings were cut out and the attached adipose tissue was immediately removed in Krebs-Henseleit solution (118.4 mM NaC1, 4.7 mM KC1, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25.0 mM NaHCO3, and 11.1 mM glucose, 37°C) (K3753; Sigma-Aldrich Japan, Tokyo, Japan).
7
Rat Aorta Tissue Extraction and Characterization
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The rats were heparinized 10–30 min before the rats were killed. After rats were anesthetized with 50 mg kg−1 IP pentobarbital and euthanized by bleeding, the thoracic aorta, from the distal aortic arch to the descending thoracic aorta at the level of the diaphragm, was dissected and immediately placed in a culture dish containing Krebs–Henseleit buffer (K3753; Sigma-Aldrich) gassed with 95% O2–5% CO2 at 37 °C. The aorta was then cleaned of surrounding connective tissue and sectioned into rings for further experiments.
Aortic sections were fixed in 10% neutral-buffered formalin or embedded in OCT compound (4583; Tissue-Tek, Tokyo, Japan) for immunohistochemical analysis, snap-frozen in liquid nitrogen for polymerase chain reaction (PCR), GLO1 activity assay, or immunoblotting, or kept in Krebs–Henseleit buffer at 37 °C under continuous bubbling with 95% O2–5% CO2 for isometric wall tension studies and measurement of NO production.
Aortic sections were fixed in 10% neutral-buffered formalin or embedded in OCT compound (4583; Tissue-Tek, Tokyo, Japan) for immunohistochemical analysis, snap-frozen in liquid nitrogen for polymerase chain reaction (PCR), GLO1 activity assay, or immunoblotting, or kept in Krebs–Henseleit buffer at 37 °C under continuous bubbling with 95% O2–5% CO2 for isometric wall tension studies and measurement of NO production.
8
Aortic Vasoreactivity Assay
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We removed the thoracic aorta after euthanasia. The vessels were dissected free of fat and connective tissue and cut into ring segments approximately 2–3 mm in width. The dissection was performed on the tension transducer in the groove full of Krebs solution (K3753, Sigma) and was maintained at 37 °C. The aortic segments pretreated with AMSC culture medium or GDNF-AMSC culture medium were equilibrated for 90 min and following which they were exposed to standard concentrations of KCl (60 Mm) for two times. KCl is washed away immediately after the vasoconstriction is stable. The aortic segments were equilibrated for 20 min; afterward, the vasoconstriction was stimulated by phenylephrine (PE) (10−6 mol/L). After the vasoconstriction was stabilized, the vasodilatation was administered sequentially with acetylcholine (Ach) at a cumulative concentration of 10−8 mol/L–10−4 mol/L and was recorded.
Vasodilatation rate = (the maximum tension before Ach administration − the tension recorded after Ach administration)/(the maximum tension before Ach administration − the basic tension) × 100%
Vasodilatation rate = (the maximum tension before Ach administration − the tension recorded after Ach administration)/(the maximum tension before Ach administration − the basic tension) × 100%
9
Cardiac Function Evaluation in Aged Mice
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After 14 weeks of dietary treatment, hearts were isolated from the old mice and perfused as previously described61 (link). Briefly, mice were lightly anesthetized by isoflurane and euthanized by cervical dislocation. The hearts were then quickly removed and placed in ice-cold buffer followed by aortic cannulation for retrograde perfusion with phosphate-free Krebs-Henseleit buffer (Sigma-Aldrich, K3753) that was supplemented with calcium chloride and sodium bicarbonate maintained at 32°C. Cardiac function was measured using a balloon placed in the left ventricle, monitored using a pressure transducer, and analyzed using the EverBeat system acquisition software (Mouse Specifics)46 (link)62 (link). Cardiac intracellular Ca2+ regulation was assessed after a 10-minute initial data acquisition period by the infusion of Ca2+ into the solution at concentrations of 1 mM, 1.5 mM, 2 mM, and 2.5 mM. Cardiac contractility was tested by infusion of 100 nM of isoproterenol into the solution.
10
Comprehensive Lung Tissue Analysis Pipeline
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After IP injection of an overdose of ketamine (300 mg/kg) and xylazine (60 mg/kg), the abdominal wall and thorax of the mice were opened to expose the heart. The mice were perfused at 100 ml/h using a multichannel syringe pump (ProSense B.V.) via the right ventricle using Krebs-Henseleit buffer (Sigma-Aldrich, K3753) containing 10 U/mL heparin (Sigma-Aldrich, H4784) to rinse away the blood.
Lung tissue was processed for several purposes, i.e., real-time quantitative PCR (RT-qPCR), nCounter digital transcriptomics profiling, flow cytometry-based immune-profiling, scanning electron microscopy, immunofluorescence microscopy and histopathological staining.
Lung tissue was processed for several purposes, i.e., real-time quantitative PCR (RT-qPCR), nCounter digital transcriptomics profiling, flow cytometry-based immune-profiling, scanning electron microscopy, immunofluorescence microscopy and histopathological staining.
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