The phrenic nerve–diaphragms were isolated as described previously [30 (link),31 (link)]. The mice were killed with CO2 exposure. Using a pair of forceps, a cotton thread was inserted under the nerve, and two knots were tied. The diaphragm was held using the lower part of the sternum to avoid damaging the muscle fibres. It was then transferred to normal Krebs solution maintained under 95% O2 and 5% CO2 at 37 °C. A cotton thread was tied (with double knots) at the apex of the triangle to connect the preparation to the transducer (Grass SD9 stimulator; Quincy, MA, USA), which was connected to a PowerLab recorder (ADIntruments, Bella Vista, NSW, Australia). The ribs at the base of the triangle were used to secure the preparation to the support. Next, the tissue was drilled between the ribs, a cotton thread was passed through, and a double knot was tied around the lower rib. The support was positioned, and the base of the triangle was tied using a double knot around the holder (Figure 2 ). Muscle twitches were evoked by indirect stimulation of the phrenic nerve for 0.05 ms at 0.2 Hz or by direct stimulation of the muscle with a pulse for 0.5 ms at 0.2 Hz. The phrenic nerve–diaphragms were equilibrated in Krebs solution and maintained under the optimal tension of 1 g for 45 min before the test.
Sd9 stimulator
Manufactured by Natus
Sourced in United States
The SD9 Stimulator is a laboratory device used to generate electrical stimuli. It produces square wave pulses that can be used to stimulate various biological preparations, such as nerve or muscle tissue. The device provides adjustable parameters, including pulse duration and frequency, to enable researchers to tailor the electrical stimuli for their specific experimental needs.
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Lab products found in correlation
Labchart software, by ADInstruments (2 mentions)
Power lab data recording system, by ADInstruments (1 mentions)
Powerlab 4 25, by ADInstruments (1 mentions)
Texas red, by Thermo Fisher Scientific (1 mentions)
Lumplfln 40xw, by Olympus (1 mentions)
A1rmp two photon microscope, by Nikon (1 mentions)
Glass bottom dish, by MatTek (1 mentions)
17 protocols using sd9 stimulator
1
Phrenic Nerve-Diaphragm Preparation Protocol
2
Papillary Muscle Electrophysiology Protocol
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The left ventricular papillary muscle was pinned between 2 earth-isolated platinum electrodes in a 1.0 mL experimental chamber filled with Tyrode's physiological salt solution (37°C, aerated with 95%O2–5%CO2). The papillary muscle was slowly stretched to a maximum preload (5–10 m⋅N) and was then stimulated using the Grass SD9 stimulator (West Warwick, RI, USA) at a frequency of 1 Hz, pulse width of 0.5 ms, and stimulus strength of 20% above threshold. After a 5 min equilibration period, the papillary muscle was then impaled by glass microelectrodes filled with 3M potassium chloride ((World Precision Instruments; Sarasota, FL, USA) filamented borosilicate glass, outer diameter 1.5 mm, tip resistance of 5–15 mΩ) using a silver/silver chloride reference electrode. The electrical activity (mV) of a cell was recorded with a Cyto 721 electrometer (World Precision Instruments) connected to a PowerLab data recording system (ADInstruments).
3
In Vivo Electroporation of Tadpole Eyes
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A 2–5 μL mix of Green fluorescent protein (GFP)-synaptobrevin and tdTomato plasmids at equimolar amounts (1 μg/μL) were loaded into an aluminosilicate glass capillary needle and mounted onto a three-axis manual micromanipulator. Tadpoles at stage 28–32 were anesthetized with 0.05% tricaine methanesulfonate and placed in an anesthetic-saturated Sylgard cushion with a custom-made trench. Tadpoles were mounted on their side, with the right eye up, using a standard size harp slice grid. A second micromanipulator holding a pair of cathode and anode copper electrodes were placed 0.1 mm apart to span the diameter of the eye. About 1–2 nL of DNA mix was pressure injected into the anterior chamber near the lens at 20 psi and 15 ms duration using a Picospritzer III pipet holder. A Grass SD9 stimulator was used to simultaneously deliver single currents of 40 V, 200 Hz, 2 ms delay, and 2 ms duration. Tadpoles recovered in fresh rearing solution immediately after electroporation. Tadpoles at stages 39–45 were screened and those with single RGCs expressing tdTomato and punctate GFP-syb in their axon terminals were selected and used for experimentation and imaging.
4
Neuromuscular Junction Electrophysiology
5
Retrograde Neuronal Labeling via Electroporation
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Texas Red (100 mg ml–1; dextran, Texas Red, 3,000 MW, lysine fixable) (ThermoFisher Scientific) in patch-clamp intracellular saline (see above) lacking ATP, GTP, biocytin and Alexa-568–hydrazide-Na was backfilled into a patch pipette. The pipette was positioned near the cell body (without any collagenase application) and two to five pulses of 10 V (2 ms duration) were applied using an SD9 stimulator (Grass Instruments). All fills and anatomy were carried out with flies on the wheel under the two-photon microscope (as in calcium imaging, except using a ×40/0.80 NA objective (LUMPLFLN 40XW, Olympus) and a 590–650 nm bandpass filter (Chroma) to filter emitted light before entering a second GaAsP detector (Hamamatsu).
6
In Vivo Imaging of Spinal Cord Neurons
7
Cardiac Muscle Contraction Modulation
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The left atrium was mounted in an organ chamber and maintained in modified Krebs–Henseleit solution (KHS) containing (in mM) 120 NaCl, 5.4 KCl, 1.2 MgCl2, 1.25 CaCl2, 11 glucose, 27 NaHCO3, and 2 NaH2PO4 (pH 7.4), oxygenated with carbogen mixture (95% O2 and 5% CO2) and maintained at 29 ± 0.1°C. The atrium was electrically stimulated (1 Hz, 80 V, 1.5 ms, SD9 stimulator, Grass). Tissue was placed under 5 mN tension, and an isometric force transducer (HP FTA 10-1 Sunborn) was used to record the contraction force. After 30 min of stabilization, MnTE-2-PyP5+ was added cumulatively to the bath (1, 3, 10, 30, 100, and 300 μM).
8
Live Cardiomyocyte Contractile Monitoring
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The cell samples on the glass slides were mounted on the AFM sample holder and loaded into the liquid cell. An illumination and detection setup similar to that in the wave propagation experiment was used to detect the cardiomyocyte contractile activities. The output of the QPDs was recorded using a National Instruments DAQ with a custom LabView 8.0 program. Sample rate was 1,000 Hz, and the filter cutoff was 100 Hz. To minimize the damage to the cells, we used soft AFM cantilevers with a spring constant in the range of 0.02 to 0.03 N/m. In the pacing experiments, the monolayered cardiomyocytes were paced with a pair of platinum electrodes with a 12-ms, 85-V pulse using an SD9 stimulator (Grass Technologies) at two pacing rates, 1.8 Hz and 4 Hz. The electrode wires were applied to the system via two channels of the AFM liquid cell. All use of experimental animals was performed according to animal use protocol (#S01013M) approved by the University of California, San Diego Institutional Animal Care and Use Committee. All use of human induced pluripotent stem cells was approved by the University of California, San Diego Human Research Protections Program and Institutional Review Board/The Embryonic Stem Cell Research Oversight Committee for project 161206ZX.
9
Rat Aortic Ring Isolation and Contractility
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The rat aortic rings were isolated according to previously described methods [28 (link)]. The rats were anaesthetised with an intraperitoneal injection of urethane (0.6 g/kg body weight) combined with chlorohydrate (0.4 g/kg body weight). Thereafter, blood was exsanguinated from the abdominal aorta. The thoracic aorta was removed and separated in normal Krebs solution (118.5 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2 2H2O, 1.2 mM KH2PO4, 1.2 mM MgSO4 7H2O, 25 mM NaHCO3, 11.1 mM glucose). The aorta was cut into 6–8-mm-long rings and subjected to an organ bath perfused with 95% O2 and 5% CO2 at 37 °C. Two “L”-type stainless steel hooks were then inserted into the aortic lumen, with one fixed to the bottom and the other connected to a force transducer. The aortic rings were maintained under the optimal tension of 1 g for 45 min (Figure 1 ). Aortic ring contraction was recorded using a force-displacement transducer (Grass SD9 stimulator; Quincy, MA, USA) that was connected to a PowerLab recorder (ADIntruments, New South Wales, Australia). The aorta rings were denuded and the endothelium was removed by rubbing with cotton; the absence of ACh-induced relaxation indicated successful denudation [28 (link)].
10
Measuring Papillary Muscle Electrical Activity
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The papillary muscle was excised from the left ventricle and a stainless steel hook inserted through the superior end. The papillary muscle was then placed between two platinum electrodes in a 1.0 mL experimental chamber filled with Tyrodes physiological salt solution (37°C; aerated with carbogen) and fixed into position with a stainless steel pin (21). The papillary muscle was then slowly stretched to a maximum pre-load (5 mN) and then was then stimulated using a Grass SD-9 stimulator and contractions were induced at 1 Hz, with a pulse width of 0.5 msec and stimulus strength 20% above threshold. After a five-minute equilibration period, the papillary muscle was then impaled by a glass electrode filled with potassium chloride 1 M (filamented borosilicate glass, outer diameter 1.5 mm, tip resistance of 5-15 mΩ when filled with 3 M KCL), using a silver/silver chloride reference electrode. The electrical activity, which was recorded in mV, of a cell was recorded following a further 25 minute equilibration period with a Cyto 721 electrometer was connected to an ADInstruments (Chart 7) recording system.
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