After the careful exposure of goat intestinal mesentery, a branch of the superior mesenteric artery adjacent to the duodenum and jejunum just before its branching into the inferior branch was dissected out and placed in cold aerated modified Krebs-Henseleit solution (MKHS) with the following composition: 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 11.9 mM NaHCO3, 1.2 mM KH2 PO4, and 11.1 mM d-glucose (pH, 7.4). Further, 1N HCl solution was added to MKHS so as to adjust the pH at 6.8 or 6.0 or 5.5 or 5.0 or 4.5.[13 (link)] Arteries were cleared of fat and connective tissues. Endothelium was removed by cotton swab method.[14 (link)] The arterial ring of 1.5–2 mm was then mounted between two fine stainless steel L-shaped hooks and kept under a resting tension of 1.5 g in a thermostatically controlled (37.0°C ± 0.5°C) automatic organ bath (Pan Lab) of 20 mL capacity containing MKHS and was aerated continuously with carbogen (95% O2+5% CO2). The arterial rings were equilibrated for 90 min before recording of muscle tension. During this period, the bathing fluid was changed for every 15 min. This experiment was repeated for both endothelium intact and denuded vessels. The change of isometric tension was measured by a high-sensitive isometric force transducer (Model: MLT0201, AD instrument, Australia) and analyzed using Chart 7.1.3 software.
Mlt0201
Manufactured by ADInstruments
Sourced in Australia
The MLT0201 is a precision temperature sensor designed for use in physiological research and monitoring applications. It features a high-accuracy thermistor housed in a compact stainless steel probe. The device is capable of measuring temperatures within a range of -50°C to 150°C with an accuracy of ±0.1°C.
Automatically generated - may contain errors
Lab products found in correlation
N12128, by ADInstruments (2 mentions)
Powerlab ml 846 data acquisition system, by ADInstruments (2 mentions)
Automatic organ bath, by Harvard Apparatus (1 mentions)
Powerlab 4 30, by ADInstruments (1 mentions)
Labchart pro, by ADInstruments (1 mentions)
Powerlab 26t, by ADInstruments (1 mentions)
Labchart 7, by ADInstruments (1 mentions)
Labchart pro software, by ADInstruments (1 mentions)
10 protocols using mlt0201
1
Goat Mesenteric Artery Tension Measurement
2
Evaluating Vascular Tone Modulation
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As described previously (Sánchez-Salgado et al. 2007 (link); Shah and Gilani 2010 (link)), the thoracic aortic rings from SD rats were used to see, specifically, effect of the extract and fractions on vascular tone. SD rats were sacrificed by cervical dislocation. Thoracic aorta was isolated, cleaned of fat and connective tissue and made into rings of 2–3 mm width. Each individual ring was carefully hooked between two stainless steel probes and suspended in a 10 mL tissue bath containing normal Kreb’s solution warmed at 37 °C and aerated with carbogen (5% CO2 in O2). The composition of Kreb’s solution was (mM): NaCl 118.2, NaHCO3 25.0, CaCl2 2.5, KCl 4.7, KH2PO4 1.3, MgSO4 1.2 and glucose 11.7 (pH 7.4). A preload of 1 g was placed on each aortic ring. Changes in isometric tension were recorded and analysed through a force transducer (MLT 0201) coupled with a bridge amplifier (N12128) and PowerLab (ML 846) Data Acquisition System (ADInstruments, Sydney, Australia). Aortic rings were allowed to equilibrate for 30–45 min. In some aortic rings, endothelium was deliberately removed by rubbing the luminal surface with forceps and were considered denuded when acetylcholine exhibited relaxation <10%. Aortic rings were pre-contracted with PE (1 µM) and effect of cumulative addition of extract and fraction was determined.
3
Colon Contractility Assay in Mice
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Colon segments (5 mm in length) were isolated from 8- to 10-week-old C57BL/6J mice and mounted on a force transducer (MLT0201, ADInstruments) connected with a PowerLab recording device (ML785, ADInstruments) for monitoring of isometric contraction. The segment was suspended in the longitudinal axis of the muscle with 0.5 g resting tension in an organ bath (37°C) containing HEPES-Tyrode (H-T) buffer (137 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl2, 1.8 mM CaCl2, 10 mM HEPES, and 5.6 mM glucose, pH 7.4) with a continuous pure oxygen supply (28 (link)). Thirty minutes after equilibration, fresh bacterial culture medium was added to the bath (5 mL H-T). Each medium was prepared by culturing a single bacterial colony for 12 hours when OD600 reached 2.5. Thirty minutes after tension recording, the bath buffer was replaced with fresh H-T solution in order to recover the segment. Usually, contractility of the segment could completely recover. When incubated with the PIB culture supernatant, the segment was recovered substantially (~50%) but not completely. This might be due to the presence of residual DPA infused into the colon tissue. For the screening experiment with jejunum, the procedures were identical, except 0.2 g resting tension was applied.
4
Investigating Vascular Calcium Regulation
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As described previously (Chan et al. 2006 (link); Shah and Gilani 2009 (link)), these protocols were specifically used to see effect of the extract and fractions on Ca2+ movements through membrane and store-operated Ca2+ channels. Rabbits were killed by a blow on the back of head; the thoracic aorta was removed and cut into rings of approximately 2–3 mm width. The tissues were suspended in normal Kreb’s solution, maintained at 37 °C, and aerated continuously with carbogen. A basal tension of 2 g was placed on each aortic ring and equilibrated for 45–60 min. Phenylephrine (1 µM) was used to stabilize the preparations. Changes in isometric tension were recorded and analysed through a force transducer (MLT 0201) coupled with a bridge amplifier (N12128) and PowerLab (ML 846) Data Acquisition System (AD Instruments, Sydney, Australia).
5
Rat Aortic Ring Contractility Assay
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Thoracic aortas were dissected from male Wistar rats (200–250 g). After the removal of connective tissue, the aorta was cut into 2–3 mm rings, which were suspended in organ baths filled with Krebs–Henseleit solution (118 mM NaCl; 4.7 mM KCl; 1.2 mM KH2PO4; 1.2 mM MgSO4; 2.5 mM CaCl2; 25 mM NaHCO3, and 11 mM glucose; pH 7.4; 37 °C) continuously gassed with carborgen gas (95% O2, 5% CO2). Aortic rings were mounted between two hooks, in which one was attached to a force transducer (MLT0201; AD Instruments, Bella Vista, New South Wales, Australia). Signals were digitalized (Power Lab 4/30; AD Instruments, Bella Vista, New South Wales, Australia) and stored on a computer for analysis using the software LabChart Pro (AD Instruments, Bella Vista, New South Wales, Australia). After an equilibrium period of 90 min under 1 g resting tension, aortic rings were contracted with phenylephrine (Phe; 10 µM), and the presence of functional endothelium was confirmed by a relaxation response to acetylcholine (ACh; 10 µM) greater than 80%. In some rings, the endothelium was mechanically removed, which was confirmed by the lack of relaxation in response to ACh [31 (link)].
6
Contractile effects of Pg. Cr on rabbit jejunum
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Rabbits were subjected to cervical dislocation. Their abdomens were opened. Their jejunums were removed and maintained in Petri dishes containing Tyrode’s solution, constantly aerated with Carbogen gas (95% oxygen/5% carbon dioxide). Portions of about 1.5 cm length of rabbits’ jejunal preparations were mounted in tissue organ baths. The tissues were stabilized in normal Tyrode’s solution for about 30 min. Following stabilization, Pg. Cr was tested on isolated rabbits’ jejunal preparations in concentrations 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 5.0 and 10.0 mg/ml [19 (link)–24 (link)]. Changes in isometric tension were recorded using force transducers (model MLT0201) coupled with bridge amplifiers FE221 connected to PowerLab 26/T (ADInstruments, Sydney, Australia). Data was recorded using Lab Chart 7 software (ADInstruments, Sydney, Australia). Intestinal responses were plotted as % of control.
7
Vasorelaxant Effects of Ellagic Acid and Sildenafil on Rat Corpus Cavernosum
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Corpus cavernosum smooth muscles/CCSMs (3 × 3 × 15 mm) were isolated from shaft of rat penile tissues under anesthesia (ketamine 60 mg/kg, xylazine 8 mg/kg; i.p.) and then rats were sacrificed by cervical dislocation. The CCSMs were washed in warm modified Krebs-Henseleit/KH solution (composition in mM: 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.5 CaCl2, 25 NaHCO3, 11 glucose) and tunica albuginea covering each CCSM was partially removed microsurgically. Each CCSM was mounted in a single channel of 4 channel organ bath (Panlab, Spain) between a steel hook at the bottom and force transducer (Model no: MLT0201; ADInstruments, Australia) at the top using two cotton threads. The tissues were maintained in KH solution at 37°C, aerated with carbogen gas (95% O2 + 5% CO2) and subjected to 500 mg of tension throughout the experiment. The tissues were washed every 15 min for 1 hour and contracted with 3 μM phenylephrine (a α1 adrenergic receptor agonist). Effects of different concentrations of ellagic acid (0.1-100 μg/mL) and sildenafil ((0.1-100 μg/mL) were observed in pre-contracted CCSMs in the interval of 6 minutes. The effects were monitored with PowerLab/8SP data acquisition system (Chart software, version 7.0; ADInstruments, Australia).[6 (link)16 (link)]
8
Aortic Ring Contractility Assay
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The procedure of Taqvi et al. [24 (link)] was followed with some modifications. Thoracic aorta was isolated from Sprague–Dawley rats of normotensive and high salt-induced hypertensive rats carefully to avoid any damage to the endothelium. The aorta was then transferred into the Kreb’s solution aerated with carbogen (5 % CO2 in O2). The composition of Kreb’s solution was (mM): NaCl 118.2, NaHCO3 25.0, CaCl2 2.5, KCl 4.7, KH2PO4 1.3, MgSO4 1.2 and glucose 11.7 (pH 7.4). It was cautiously cleaned off fats and other connective tissues and then cut into rings 2–3 mm wide. In some rings, the endothelium was intentionally removed by gentle rubbing of the intimal surface with forceps. The rings with intact endothelium that produced less than 80 % relaxation in response to acetylcholine (1 µM) were tossed away. Individual rings were suspended in 10 mL tissues baths at 37 °C aerated with carbogen. A preload of 1 g was applied to each preparation and incubated for 30 min. Changes in isometric tension were recorded and analyzed through a force transducer (MLT 0201) coupled with a bridge amplifier (N12128) and PowerLab (ML 846) Data Acquisition System (ADInstruments).
9
Aortic Vasodilation Mechanism Assessment
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The thoracic portion of aorta was dissected and cut into rings of 2-3 mm. Aortic rings were placed in 10 mL organ chambers containing Krebs-Henseilet solution (in mM: NaCl 118; KCl 4.7; KH 2 PO 4 1.2; MgSO 4 1.2; CaCl 2 2.5; NaHCO 3 25; glucose 11; pH 7.4), continuously gassed with carbogen gas (95% O 2 / 5% CO 2 ) and maintained at 37 ± 0.5 °C. Isometric tension was measured (MLT0201; ADInstruments, Bella Vista, New South Wales, AUS), recorded and analyzed by using LabChart Pro software (ADInstruments, Bella Vista, New South Wales, AUS). After an equilibration period of 90 min, under 9.81 mN tension, the concentration-response curve for phenylephrine (Phe; 10 -9 to 10 -5 M; Sigma-Aldrich, St. Louis, MO, USA) was performed. When the contractile response to Phe reached a plateau (10 -5 M), the concentration-response curve for acetylcholine (ACh; 10 -9 to 10 -5 M; Sigma-Aldrich, St. Louis, MO, USA) was obtained. The response to ACh was expressed as the percentage relaxation of the maximal contraction induced by Phe (Leão et al. 2015) .
10
In Vitro Duodenal Contractility
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Duodenum segments (0.6 cm long) were dissected and processed to investigate in vitro contractility (Araujo-Neto et al. 2010). Brie y, the duodenal strips were placed in Petri dishes containing cold modi ed Tyrode's solution (composition in mM: NaCl, 136.0, KCl, 5.0, MgCl 2 , 0.98, CaCl 2 , 2.0, NaH 2 PO 4 , 0.36, NaHCO 3 , 11.9, and glucose, 5.5). Then, each segment was placed in a glass organ bath containing 5 mL of Tyrode's solution at 37°C and continuously bubbled with carbogen (5% CO 2 and 95% O 2 ) to maintain the pH 7.4. The lower end of the strip was fastened to a xed hook at the bottom of the organ bath, and the upper end was connected with a steel hook to a force transducer (model MLT0201, AD Instruments, BellaVista, NSW, Australia) under 1-g baseline tension. A concentration-response curve to acetylcholine was generated using increasing and cumulative concentrations ranging from 1 × 10 -10 to 1 × 10 -4 M, which were added into the organ bath every 5 min. The data registered from the cholinergic concentration-response curve were analyzed as the percentage contractile response compared to the mean of two standard contractions of 60 mM KCl.
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