SAA with a purity of >98% by HPLC analysis was provided by the Institute of Materia Medica (Beijing, China). SB431542, Hoechst 33342, monocrotaline (MCT) and 2',7'-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma-Aldrich (Shanghai, China). Antibodies against CD31, α-SMA, HO-1, Nrf2, 3-nitrotryosine, TGFβ1 and Nox4 were purchased from Abcam (Cambridge, UK). Antibodies against p-Smad2/3, CD68, RhoA, p-Cdc42, Cdc42, p-Cofilin, p-Smad1/5 and eNOS were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against GAPDH and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Monocrotaline mct
Manufactured by Merck Group
Sourced in United States, Sao Tome and Principe
Monocrotaline (MCT) is a chemical compound used in laboratory settings. It is a pyrrolizidine alkaloid that can be used as a tool for research purposes. MCT has specific chemical and biological properties that make it useful in certain experimental contexts, but a detailed description of its core function would require more information to provide an accurate and unbiased account while maintaining conciseness.
Automatically generated - may contain errors
Lab products found in correlation
Sprague dawley rat, by Charles River Laboratories (2 mentions)
α sma, by Abcam (1 mentions)
2 7 dichlorofluorescin diacetate dcfh da, by Merck Group (1 mentions)
Antibodies against gapdh and β actin, by Santa Cruz Biotechnology (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Sb431542, by Merck Group (1 mentions)
Antibodies against cd31, by Abcam (1 mentions)
Antibodies against p smad2 3, by Cell Signaling Technology (1 mentions)
P cofilin, by Cell Signaling Technology (1 mentions)
Tgf β1, by Abcam (1 mentions)
P smad1 5, by Cell Signaling Technology (1 mentions)
Cdc42, by Cell Signaling Technology (1 mentions)
Anti mir control, by Horizon Discovery (1 mentions)
Alda 1, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Baicalin, by Merck Group (1 mentions)
Anti mmp2, by Abcam (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Colorimetric tunel apoptosis assay kit, by Beyotime (1 mentions)
28 protocols using monocrotaline mct
1
Pulmonary Hypertension Mechanistic Insights
2
Monocrotaline-Induced Pulmonary Hypertension
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Monocrotaline (MCT) was obtained from Sigma. Anti-miR-138-5p and anti-miR-Control were obtained from Dharmacon.
3
Alda-1 Ameliorates Monocrotaline-Induced PAH
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All animal care and experimental procedures were approved and conducted in accordance with the Institutional Animal Care and Use Committee of Jinzhou Medical University and conformed to the Guide for the Care and Use of Laboratory Animal published by the US National Institutes of Health. Male Sprague–Dawley rats (n=48; weighing 220–250 g) were purchased from Vital River Laboratories Animal Company (Beijing, China). The animals were intraperitoneally (i.p.) injected with a single dose of monocrotaline (MCT; 60 mg/kg; Sigma-Aldrich, St. Louis, MO) to induce severe PAH within 2 or 4 weeks (n=8 each group). For experiments involving pre-treatment with Alda-1 (Sigma-Aldrich Co., St. Louis, MO), MCT-injected rats were randomly divided into 3 groups, including the MCT group (n=12), the vehicle-alone group (n=6) administered 50% polyethylene glycol (PEG) and 50% dimethyl sulfoxide (DMSO) by volume, and the Alda-1 group (n=6). Control rats (n=8) were injected with an equal volume of 0.9% phosphate-buffered saline (PBS). The MCT-treated rats were subcutaneously implanted with mini-osmotic pumps (model 2004; ALZET, Cupertino, CA) and continuously infused with Alda-1 (10 mg kg−1 d−1) for 4 weeks.
4
Baicalin Attenuates Monocrotaline-Induced Pulmonary Hypertension
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Male Wistar rats weighing 200–250 g were purchased from animal center of Second Hospital of Shandong University. Male rats were used to minimize hormonal effects (e.g., of estrogen). The animal protocols followed the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Shandong University. All rats received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health.
Baicalin (purity >95%) was purchased from Sigma and was dissolved in dimethyl sulfoxide (DMSO, Sigma‐Aldrich). PAH model was induced by intraperitoneal injection of 50 mg/kg of monocrotaline (MCT) for 6 weeks (Sigma‐Aldrich) as per our previously described with modifications.16 Blood samples were collected at specific time points, the plasma concentration was determined,18 and we choose the drug concentration according to the report.15 In the present study, we have tested the lower dose (20 mg/kg, 50 mg/kg) and higher dose (200 mg/kg), the most effective dose is 100 mg/kg, and the main purpose of this present study is to explore the mechanism, so we only choose 100 mg/kg. Baicalin or the same amount of saline solution was given by intragastric administration from 2 days after MCT injection. Forty animals were randomly assigned to four groups: Control, MCT, saline‐, and Baicalin‐treated groups (n = 10 in each).
Baicalin (purity >95%) was purchased from Sigma and was dissolved in dimethyl sulfoxide (DMSO, Sigma‐Aldrich). PAH model was induced by intraperitoneal injection of 50 mg/kg of monocrotaline (MCT) for 6 weeks (Sigma‐Aldrich) as per our previously described with modifications.
5
Monocrotaline-Induced Rodent PH Model
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Sprague-Dawley rats (SPF, male, 180–200 g, 4 weeks) and C57/BL6 mice (SPF, male, 25–30 g, 4 weeks, WT mice) were obtained from the Animal Experimental Center of Zhejiang University, China. H19−/− mice (SPF, male, 25–30 g, 4 weeks) were gifts from Luisa Dandolo [35 (link)]. Rats received one subcutaneous (sc) injection of 60 mg/kg monocrotaline (MCT) (Sigma Chemicals, St. Louis, MO, USA) and grouped as follows: control (n = 6), sc injection of the vehicle saline (0.1 mL/kg); MCT (n = 6), sc injection of MCT [36 (link)]. C57/BL6 and H19−/− mice were given a sc injection of MCT at 600 mg/kg once a week for 8 consecutive weeks, and the mice were grouped as above [37 (link)]. Rats or mice were isoflurane-anesthetized and sacrificed after 3 or 8 weeks, respectively. Lung and heart tissues were removed, immediately frozen in liquid nitrogen and fixed in 4% buffered paraformaldehyde solution. All experimental procedures were conducted in agreement with the principles approved by the Institutional Animal Care and Use Committee of Zhejiang University.
6
Monocrotaline-Induced Pulmonary Hypertension
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Sprague-Dawley rats, male, were retrieved from the Experimental Animal Center of Zhejiang Province. The study protocol was authorized by the Ethics Review Board of Animal Use Application of The Lishui Municipal Central Hospital (Lishui, China) in compliance with the National Institutes of Health Guidelines For the Care and Use of Experimental Animals. Prior to the experiments, the rats were placed in a room under the controlled conditions of 20–26°C, 45–55% humidity, a 12 h light/dark cycle, and freely available water and standard rat chow.
SSE (purity>99.0%), bovine serum albumin (BSA) and monocrotaline (MCT) were obtained from Sigma Aldrich (Darmstadt, Germany). SSE was dissolved in normal saline. MCT was liquefied in 1M HCL neutralized with 1M NaOH, and thinned down with standard saline. Thereafter, PH was altered to 7.2–7.4. Anti-PCNA, anti-CyclinD1, anti-MMP2, anti-MMP9, anti-Bax, and anti-Bcl-2, antibodies were purchased from Abcam (Cambridge, United Kingdom). Anti-Cleaved Caspase-3, anti-p-GSK3β, anti-GSK3β, anti-β-catenin, anti-p-AKT, anti-AKT, anti-p-ERK, anti-ERK and anti-GAPDH were retrieved from Cell Signaling Technology (Danvers, MA, United States). The hematoxylin and eosin (H&E) stain kit was obtained from Solarbio (Beijing, China). The Colorimetric TUNEL Apoptosis assay kit was purchased from Beyotime Institute of Biotechnology (Jiangsu, China).
SSE (purity>99.0%), bovine serum albumin (BSA) and monocrotaline (MCT) were obtained from Sigma Aldrich (Darmstadt, Germany). SSE was dissolved in normal saline. MCT was liquefied in 1M HCL neutralized with 1M NaOH, and thinned down with standard saline. Thereafter, PH was altered to 7.2–7.4. Anti-PCNA, anti-CyclinD1, anti-MMP2, anti-MMP9, anti-Bax, and anti-Bcl-2, antibodies were purchased from Abcam (Cambridge, United Kingdom). Anti-Cleaved Caspase-3, anti-p-GSK3β, anti-GSK3β, anti-β-catenin, anti-p-AKT, anti-AKT, anti-p-ERK, anti-ERK and anti-GAPDH were retrieved from Cell Signaling Technology (Danvers, MA, United States). The hematoxylin and eosin (H&E) stain kit was obtained from Solarbio (Beijing, China). The Colorimetric TUNEL Apoptosis assay kit was purchased from Beyotime Institute of Biotechnology (Jiangsu, China).
7
Monocrotaline-Induced Cardiac Dysfunction
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Male Wistar rats of 305 ± 4 g (mean ± SEM, n = 10) were subcutaneously injected with 60 mg kg−1 (body weight) monocrotaline (MCT, Sigma Aldrich, Australia) at 57 ± 1 days of age. Aged-matched control rats (CON) of 310 ± 3 g, n = 11, were injected with the same volume of sterile saline. Animals were fed normal rat chow and water ad libitum for 4 weeks and monitored regularly. At 28–32 days post injection, rats were anaesthetized using 2% isoflurane in 100% O2 as a carrier gas and in vivo electrocardiogram recordings performed. Approval for this research was provided by the University of Auckland Animal Ethics Committee (AEC: 001807), in accordance with the Code of Ethical Conduct of The University of Auckland, and the New Zealand Animal Welfare Act 1999.
8
Rat Pulmonary Hypertension Induction
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All experiments conducted in this study were approved by the Federal Ministry of Science, Research and Economy (6609/196-II/10 b/2008). A total of 62 male Sprague-Dawley rats, weighing 350–500 g, underwent left-sided unilateral pneumonectomy and simultaneous implantation of a telemetry catheter into the common pulmonary artery trunk. This technique was developed and refined by our group (manuscript under review).
Animals were kept at room temperature of 22℃ and 12:12 h day-night cycle. They were provided tap water and common rat chow (ssniff-RMH, Soest, Germany) ad libitum. Seven days after surgical intervention, PAH was induced by subcutaneous administration of 60 mg/kg monocrotaline (MCT; Sigma, Vienna, Austria).
Animals were kept at room temperature of 22℃ and 12:12 h day-night cycle. They were provided tap water and common rat chow (ssniff-RMH, Soest, Germany) ad libitum. Seven days after surgical intervention, PAH was induced by subcutaneous administration of 60 mg/kg monocrotaline (MCT; Sigma, Vienna, Austria).
9
MCT-Induced Pulmonary Hypertension Model
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It is well recognized that MCT treatment is one of the most popular method for creating a validated animal model of PAH.45 , 46 The procedure and protocol were based on our previous reports.45 , 46 In detail, by Day 0, pathogen‐free, adult male Sprague–Dawley (SD) rats, weighing 325–350 g (Charles River Technology, BioLASCO Co., Ltd., n = 24) were given one subcutaneous injection of monocrotaline (MCT) (63 mg/kg; Sigma). On Day 5, these MCT‐treated animals were assigned to two experimental groups: group 2 (MCT alone, n = 8) and group 3 (MCT + 0.1% PTU [Sigma] in drinking water daily, n = 8). Another group 1 (i.e. sham‐operated control [SC], n = 8) that receiving only subcutaneous injection of 3 ml normal saline served as SC. PTU therapy was implemented immediately after the assignment. The dosage of MCT utilized here was based on our previous reports45 , 46 with minimal modification of MCT dosage.
10
HUVEC Cell Culture and Protein Analysis
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Human umbilical vein endothelial cells (HUVECs) (BCRC H‐UV001) at passages 3–6 were cultured in M199 medium (Gibco) that contained 20% Fetal Bovine Serum (Hyclone), 1% ECGS (Millipore), 0.2% Heparin (China Chemical & Pharmaceutical Co., Ltd), 100 μg/ml streptomycin and 100 units/ml penicillin (Gibco). In detail, 1 × 106 HUVEC cells were seeded in a 10‐cm dish. After overnight incubation, cells were treated with 10 ng/ml of transforming growth factor (TGF)‐β1 (R&D), 2 μM of monocrotaline (MCT) (Sigma) or 20 ng/ml propylthiouracil (PTU) (Panbiotic Laboratories Co., Ltd.). After additional incubation for 48 h, the cells were collected, and protein extract was harvested with RIPA buffer for Western blot analysis.
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