Shortly before the experiments, an aliquot of cell suspension that contained GH3 cells or Rolf B1.T olfactory neurons, were gently harvested and transferred immediately to a home-made recording chamber which was firmly positioned on the stage of a CKX-41 inverted microscope (Olympus; YuanLi, Kaohsiung, Taiwan). Cells were immersed at room temperature (22–25 °C) in normal Tyrode’s solution, the composition of which is detailed above. Patch clamp recordings under the whole-cell mode were applied with either an RK-400 (Biol-Logic, Claix, France) or an Axopatch-200B amplifier (Molecular Devices; Bestgen Biotech, New Taipei City, Taiwan) [41 (link),59 (link)]. Patch electrodes with tip resistances of 3–5 MΩ were made of Kimax-51 glass capillaries (#34500; Kimble; Dogger, New Taipei City, Taiwan) on either a PP-83 vertical puller (Narishige; Major Instruments, New Taipei City, Taiwan) or a p-97 horizontal puller (Sutter, Novato, CA), and then fire-polished with MF-83 microforge (Narishige). Spontaneous ACs were measured by using cell-attached voltage clamp recordings, and the potential was held at the level of the resting potential (around −70 mV) [62 (link)].
Pp 83 vertical puller
Manufactured by Narishige
Sourced in Canada, United Kingdom, Japan, United States
The PP-83 is a vertical glass micropipette puller manufactured by Narishige. It is designed to create glass micropipettes for use in various laboratory applications. The device utilizes a heat-based pulling mechanism to draw out and shape glass capillaries into fine-tipped micropipettes.
Automatically generated - may contain errors
Lab products found in correlation
Mf 83 microforge, by Narishige (3 mentions)
Ckx41 inverted microscope, by Olympus (3 mentions)
Rk 400, by Bio-Logic (3 mentions)
Axopatch 200b amplifier, by Molecular Devices (3 mentions)
P 97 horizontal puller, by Sutter Instruments (2 mentions)
Dm il inverted microscope, by Leica camera (2 mentions)
Axopatch 200b, by Molecular Devices (2 mentions)
Axoclamp 2b, by Molecular Devices (2 mentions)
Axopatch 200b patch amplifier, by Molecular Devices (1 mentions)
Axopatch 200a amplifier, by Molecular Devices (1 mentions)
Rk 400 amplifier, by Bio-Logic (1 mentions)
Borosilicate glass, by Narishige (1 mentions)
7 protocols using pp 83 vertical puller
1
Whole-cell Patch-clamp Electrophysiology
2
Patch-Clamp Whole-Cell Recordings of GH3 or Jurkat T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly before the recordings, we harvested GH3 or Jurkat T cells and rapidly resuspended an aliquot of cell suspension to a custom-made cubicle mounted on the fixed stage of CKX-41 inverted microscope (Olympus; YuanLi, Kaohsiung, Taiwan). We the immersed cells at room temperature (20–25°C) in normal Tyrode’s solution, the composition of which has been described above in detail. We exploited either a P-97 Flaming/Brown horizontal puller (Sutter Instruments, Novato, CA) or a PP-83 vertical puller (Narishige; Taiwan Instrument, Taipei, Taiwan) to fabricate the recording pipette electrodes, which were made of Kimax-51 glass capillaries (Kimble; Dogger, New Taipei City, Taiwan), and we then fire-polished electrode tips with an MF-83 microforge (Narishige). The patch electrodes, in which different internal solutions were filled up, had a tip resistance of 3 to 5 MΩ. In this study, we undertook standard patch-clamp whole cell recordings at room temperature by applying either an RK-400 (Bio-Logic, Claix, France) or an Axopatch-200B patch-amplifier (Molecular Devices, Sunnyvale, CA). To measure whole-cell data, the junctional voltage between the pipette and bath solution was set as zero once the electrode was bathed but shortly before the giga-seal (>1 GΩ) formation. The details of data recordings and analyses achieved in the present work were described in Supplementary Material
3
Patch-Clamp Analysis of Smooth Muscle Cell Ion Currents
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell Kv and KATP currents were recorded from single smooth
muscle cells using the patch clamp technique. Currents were filtered at 2 kHz (-3 dB) and
recorded with an Axopatch 200A amplifier (Molecular Devices) and digitized at 10 kHz.
Patch pipettes were made from thick-walled borosilicate glass (Clark Electromedical,
Pangbourne, Berks, UK) using a pp-83 vertical puller (Narishige, Tokyo, Japan). Electrode
resistances before sealing were 3–5 MΩ and after sealing were >1 GΩ . All experiments
were done at 30 °C, maintained using a Dagan HW-30 temperature controller.
muscle cells using the patch clamp technique. Currents were filtered at 2 kHz (-3 dB) and
recorded with an Axopatch 200A amplifier (Molecular Devices) and digitized at 10 kHz.
Patch pipettes were made from thick-walled borosilicate glass (Clark Electromedical,
Pangbourne, Berks, UK) using a pp-83 vertical puller (Narishige, Tokyo, Japan). Electrode
resistances before sealing were 3–5 MΩ and after sealing were >1 GΩ . All experiments
were done at 30 °C, maintained using a Dagan HW-30 temperature controller.
4
Patch-Clamp Recordings of GH3 and PC12 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
On the day of the experiments, cells (i.e., GH3 or PC12 cells) were harvested and a few drops of cell suspension was immediately transferred to a home-made recording chamber affixed to the stage of a DM-IL inverted microscope (Leica, Wetzlar, Germany). Cells visualized under inverted microscope were immersed at room temperature in normal Tyrode’s solution, the composition of which is detailed above. We performed the patch-clamp recordings under whole-cell configuration with either an RK-400 (Biologic, Echirolles, France) amplifier, or an Axopatch-200B or Axoclamp-2B (Molecular Devices, Sunnyvale, CA, USA) amplifier [20 (link)]. The recording pipette electrodes with tip resistances of 3–5 MΩ were prepared from Kimax-51 glass capillaries (#34500 (outer diameter: 1.5–1.8 mm); Kimble Products, Vineland, NJ, USA), and we fabricated the capillary tubes by using either a PP-83 vertical puller (Narishige, Tokyo, Japan) or a P-97 horizontal puller (Sutter, Novato, CA, USA). An electrode holder filled with a silver chloride-coated silver wire connected the patch electrode to the amplifier. During the measurements, the electrode used was mounted on and delicately controlled by a WR-98 hydraulic micromanipulator (Narishige).
5
Patch-clamp Whole-cell Recordings
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Shortly before the experiments, cells were harvested and an aliquot of cell suspension was transferred to a home-made recording chamber mounted on the fixed stage of CKX-41 inverted microscope (Olympus, Tokyo, Japan). Cells were bathed at room temperature (20–25 °C) in normal Tyrode’s solution, the composition of which was indicated above. The recording electrodes were made of Kimax-51 glass capillaries (Kimble, Vineland, NJ, USA) by using either a PP-83 vertical puller (Narishige, Tokyo, Japan) or a P-97 Flaming/Brown horizontal puller, and their tips were then fire-polished with an MF-83 microforge (Narishige; London, UK). The electrodes used had a resistance of 3–5 MΩ and were filled with different internal solutions as detailed above. Patch-clamp whole cell recordings were performed at room temperature in standard patch-clamp technique by use of either an RK-400 amplifier (Bio-Logic, Claix, France), or an amplifier of Axopatch-200B or Axoclamp-2B (Molecular Devices, Sunnyvale, CA, USA). The junction potential between the pipette and bath solution was nulled after the pipette entered the bath but immediately before seal formation was made, and whole-cell data were hence corrected.
6
Whole-cell Current-clamp Recordings of Adiponectin Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell current-clamp recordings were performed as previously reported47 (link) Briefly, pipettes were used with 3–9 MΩ resistance after being filled with pipette solution. Pipettes were made of borosilicate glass (Narishige) using a PP-83 vertical puller (Narishige) or a Sutter micropipette puller (P-1000). The pipettes were used with 3–9 MΩ resistance after being filled with pipette solution. The composition of the pipette solution was as follows (in mM): 135 K-gluconate, MgCl2 2, HEPES 10, EGTA 1.1, Mg-ATP 2.5, Na2-GTP 0.3, and Na2-phosphocreatine 10, pH 7.3 with KOH (with an osmolarity of 290–295 mOsm). An Axopatch 200B amplifier and Clampex 9.2 or 10 software (Axon Instruments) were used for data acquisition. Pclamp 9.2 or 10 (Axon Instruments) software was used for analysis. Liquid junction potential correction was performed off-line. Access resistance was continuously monitored during the experiments. Only those cells in which access resistance was stable (changes ~30%) were included in the analysis. The data was analyzed by Clamp fit 9.2 or 10 (Axon instruments) software and GraphPad Prism6 software. The response by adiponectin was defined as a change over 2 times standard deviation of membrane potential for 2 min before applying adiponectin.
7
Patch-clamp electrophysiology on adherent cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Before the experiments, we gently dispersed cells with a 1% trypsin/EDTA solution, and an aliquot of cell suspension was directly placed in a recording chamber attached to the fixed-stage of a DM-IL inverted microscope (Leica; Highrise Instrument, Taichung, Taiwan). Cells were immersed at room temperature (20–25 °C) in normal Tyrode’s solution containing 1.8 mM CaCl2. The electrodes that we used were fabricated from Kimax-51 capillaries (Merck, Taipei, Taiwan) using a PP-83 vertical puller (Narishige; Taiwan Instrument, Tainan, Taiwan), and their tips were thereafter fire-polished with an MF-83 microforge (Narishige; Taiwan Instrument, Tainan, Taiwan). As the electrodes were filled with the different internal solutions described above, their resistance was measured to range between 3 and 5 MΩ, for the purpose of avoiding excessive damage to the cell. Patch-clamp recordings were carried out in whole-cell configuration using either an RK-400 (Bio-Logic, Claix, France) or an Axopatch-200B amplifier (Molecular Devices; Bestgen Biotech, New Taipei City, Taiwan), as described elsewhere [31 (link),37 (link),52 (link),67 (link)]. Whole-cell recording was achieved by rupturing the patch of membrane isolated with GΩ sealing by the patch pipet, which brings the cell interior into contact with the pipet interior.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!