After injection with 50 nl TRPV2 cRNA (500 ng/μl), oocytes were incubated at 17 °C for 4–6 days. Ionic currents were recorded using an OC-725C amplifier (Warner Instruments) and digitized using Digidata 1440 (Axon Instruments). The recording bath solution contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2 and 5 mM HEPES at pH 7.4 (adjusted with NaOH). Oocytes were voltage-clamped at −60 mV, and current data was obtained every 1 s. Chloramine T (ChT; MP Biomedicals) was dissolved in the bath solution. For heat stimulation, bath solution heated with a temperature controller (TC-344B; Warner Instruments) was applied by perfusion. For poking assay, mechanical stimulation was applied using a tungsten needle (outer diameter, 40 μm) mounted on the manual manipulator (NARISHIGE NMN-21). The needle set to 40° from the horizontal plane into the moving parts of manipulator. The tip of the needle was positioned through the rotation of dials so that it just attached to the cell membrane. After that, we rotated the dials (Z-axis) of manipulator (2.5 rotation). The needle was then moved toward the oocyte as the 8 μm step. It takes for 30 s that the needle moved 8 μm.
Nmn 21
Manufactured by Narishige
The NMN-21 is a manual micromanipulator produced by Narishige. It is designed for precise control and positioning of microelectrodes and other small instruments during microscopic procedures. The NMN-21 provides three-dimensional movement with micrometer-level control.
Automatically generated - may contain errors
Lab products found in correlation
Axopatch 200b, by Molecular Devices (2 mentions)
Female crimp terminal, by Narishige (2 mentions)
Silver wire, by Merck Group (2 mentions)
Low melt agarose, by Merck Group (2 mentions)
Male crimp terminals, by RS Components (2 mentions)
Female crimp terminal, by RS Components (2 mentions)
Digidata 1440, by Molecular Devices (1 mentions)
Oc 725c amplifier, by Warner Instruments (1 mentions)
Tc 344b, by Warner Instruments (1 mentions)
Te2000 u, by Nikon (1 mentions)
Plan fluor elwd 40, by Nikon (1 mentions)
Agarose, by Merck Group (1 mentions)
Naclo, by Merck Group (1 mentions)
Digidata 1440a, by Molecular Devices (1 mentions)
Low gelling agarose, by Merck Group (1 mentions)
Clampfit, by Molecular Devices (1 mentions)
8 protocols using nmn 21
1
TRPV2 Ion Channel Characterization
2
Patch-Clamp Recordings in Faraday Cage
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Electrical recordings were conducted inside a Faraday cage, which contained a stage (Mechanical Workshop, University of Oxford) supporting the headstage of a conventional patch-clamp amplifier (Axopatch 200B, Axon Instruments) and two micromanipulators (Narishige, NMN-21) to guide the Ag/AgCl electrodes. The PMMA chamber, containing lipid-in-oil, was placed at the center of the stage.
3
Optical Trapping and Manipulation of Hollow Microcapsules
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The OT system (MMS, Sigma Koki) is composed of an inverted optical microscope (TE2000-U, Nikon) with an objective lens (Plan Fluor ELWD 40 ×/0.60, Nikon), a cw Nd:YAG laser (λ = 1064 nm), and other optical devices including a CCD camera (Retiga Exi, Q-Imaging) and a fast CMOS camera (PL-A741, Pixelink). The microscope stage was heated with a silicone rubber electric heating sheet and was maintained at either 25°C or 60°C using a temperature controller (DB1000, CHINO). The light intensity at the focal point ranged from 5 mW to 300 mW, and the longest laser emission duration was 250 seconds. To manipulate the hollow microcapsules, we used a tungsten microneedle with a tip diameter of 500 nm under the control of a patch clamp micromanipulator (NMN-21, Narishige).
4
Fabrication of Ag/AgCl Electrodes
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Silver wires (100 μm diameter, Sigma) were soldered into male crimp-terminals (RS Components). The tips of the wires were incubated in sodium hypochlorite, NaClO (10% active chlorine, Sigma) for 1 h to form Ag/AgCl electrodes. The tips of the electrodes were coated with a hydrogel layer by pipetting melted agarose (1% low-melt agarose, Sigma) onto their surfaces. For single DIB experiments, each electrode was plugged into a female crimp-terminal, which was attached to a micromanipulator (Narishige, NMN-21). The other end of the female crimp-terminal (RS Components) was soldered to a cable that terminated with a male crimp, which was connected to the headstage of the amplifier.
5
Electrical Recordings of Droplet Networks
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Electrical recordings were conducted with a patch-clamp amplifier (Axopatch 200B, Axon Instruments) with its head stage, contained in a Faraday cage, connected to two Ag/AgCl wire electrodes (Sigma) 100 μm in diameter. The tips of both electrodes were coated with a hydrogel at 0.5% (w/v; low gelling agarose; Sigma). The electrodes were then inserted in droplets or placed on the sides of networks with the aid of micromanipulators (NMN-21, Narishige). A current signal was obtained by episodic acquisition and filtered at 2 kHz. Data were collected at 10 kHz at an interval of 100 μs and ×5 gain with a Digidata 1440A digitizer (Axon Instruments). Using an analog output, a waveform epoch with steps at 0, 50, 0, −50, and 0 mV, each with a duration of 10 s, was used to detect the current between droplet pairs or through networks. Data were analyzed by Clampfit (version 10.3; Axon Instruments), filtering with a low pass Bessel (8 pole), and −3-dB cutoff of 40 Hz. All electrical recordings were conducted at 22.0 ± 1.5°C.
6
Intracranial injection of GEVI AAV in mice
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Adeno-associated viruses containing the sequence of GEVIs of interest were mixed with trypan blue solution, and loaded into a Hamilton syringe, attached to a Narishige mechanical micromanipulator (NMN-21). Newborn (P0.5–P1) mice of either sex were cold anesthetized by placing on ice for a couple of minutes, and then positioned on the pad below the Hamilton syringe, so that the needle touches the skull surface at a location approximately 0.25 mm lateral to the sagittal suture and 0.50–0.75 mm rostral to the neonatal coronary suture. The needle was then carefully inserted into the skull 2–3 mm deep via a micromanipulator. A volume of 1–2 μl of solution was slowly injected (for ∼30 s with several 3–5 s pauses) into the lateral ventricle. After the injections, bright white light was shone through the skull to reveal trypan blue-filled ventricles, and mice were placed on a heated pad to recover prior to returning them to the breeding cage.
7
Reconfiguring Bilayer-based Bio-pixels
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Bio-pixels were formed from two droplets in 7 mM DPhPC in hexadecane:silicone oil (1:1). The bilayer area was altered by pulling the droplets apart or pushing the droplets together with a micromanipulator (Narishige, NMN-21). The pixels were illuminated and the current profiles recorded.
8
Fabrication of Ag/AgCl Electrodes
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Silver wires (100 μm diameter, Sigma) were soldered into male crimp-terminals (RS Components). The tips of the wires were incubated in sodium hypochlorite, NaClO (10% active chlorine, Sigma) for 45 min to form Ag/AgCl electrodes. The tips of the electrodes were coated with a hydrogel layer by pipetting melted agarose (2% low-melt agarose, Sigma) onto their surfaces. Each electrode was plugged into a female crimp-terminal, which was attached to a micromanipulator (Narishige, NMN-21). The other end of the female crimp-terminal (RS Components) was soldered to a cable that terminated with a male crimp, which was connected to the voltage-clamp amplifier (Triton+, Tecella LLC) via an electrode probe holder (Terrapin, Tecella LLC). The current signal was obtained by an episodic acquisition routine (0 mV, 50 mV, 0 mV, −50 mV, 0 mV, in 5 s intervals) with a feedback resistor of 1 GΩ. Data were acquired at 20 kHz with a 5 kHz filter through the ‘TecellaLab v0.90 type 2’ software. The data was in Tecella’s tlc binary file type and analysed and displayed by using custom software written in LabVIEW31 (link).
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