Two‐electrode voltage clamp was performed with an OC‐725C oocyte clamp (Warner Instruments, Hamden, CT, USA) controlled by pCLAMP10.2 (Molecular Devices, San Jose, CA, USA). Electrodes were pulled from thin‐walled borosilicate glass capillaries with outer diameter of 2.0 mm and inner diameter of 1.56 mm (Sutter Instruments, catalogue no. BF100‐78‐10, Navato, CA, USA) and filled with 3 m KCl. The electrodes had a resistance of 0.5−2.0 MΩ. For voltage clamp recording, an oocyte was placed in a chamber containing standard ND96 solution and impaled with two microelectrodes, one for sensing membrane potential (Vm) and the other for passing current. The cell was superfused with ND96 until the Vm was stable. The current−voltage (I‐V) relationship was generated by voltage clamp using a protocol stepping the potential from −160 mV to +60 mV with 20‐mV increments. Each step was recorded for a duration of 100 ms followed by an interval of 100 ms at a holding potential close to the spontaneous Vm of the oocyte. The I‐V relationship for CO2/HCO3− was generated immediately after the hyperpolarization was maximized upon the switch from ND96 to 5% CO2/33 mm HCO3−.
Bf100 78 10
Manufactured by Sutter Instruments
Sourced in United States, Japan
The BF100-78-10 is a borosilicate glass capillary that is suitable for micropipette fabrication. It has an outer diameter of 1.0 mm and an inner diameter of 0.78 mm. The capillary is 10 cm in length.
Automatically generated - may contain errors
4 protocols using bf100 78 10
1
Two-Electrode Voltage Clamp of Oocytes
2
Establishing Wolbachia Infection in Drosophila nigrosparsa
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Cytoplasm containing a single Wolbachia strain from Drosophila melanogaster (either wMel provided by Luis Teixeira, wMelCS, or wMelPop provided by Francis M. Jiggins and Julien Martinez) was injected into the posterior end of dechorionated embryos of the uninfected D. nigrosparsa line nu_0 at Generation 0 using a micromanipulator (M‐152, Narishige) with a capillary (BF100‐78‐10, Sutter Instrument) attached to an inverted microscope (CKX53, Olympus). Injected embryos were placed on grape juice agar plates with fresh blobs of yeast and transferred into an incubator (MLR‐352H‐PE, Panasonic) for 2 days at 19°C. Injected embryos developed on malt food. Each surviving female adult was mated with an uninfected male from line nu_0 to generate infected fly lines, and each mating pair was kept separately in the mating cage. Three stably infected lines were generated, that is, ni_3, ni_6, and ni_8. To check for Wolbachia infection, five D. nigrosparsa females per line were randomly killed in each generation, and DNA was extracted and checked for infection by PCR using wsp81F and 691R primers.
3
Transinfection of Drosophila nigrosparsa
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Cytoplasm containing a single Wolbachia strain from Drosophila melanogaster (either wMel provided by Luis Teixeira, wMelCS, or wMelPop provided by Francis M. Jiggins and Julien Martinez) was injected into the posterior end of dechorionated embryos of the uninfected D. nigrosparsa line nu_0 at Generation 0 using a micromanipulator (M-152, Narishige, Japan) with a capillary (BF100-78-10, Sutter Instrument, USA) attached to an inverted microscope (CKX53, Olympus, Japan). Injected embryos were placed on grape-juice agar plates with fresh blobs of yeast and transferred into an incubator (MLR-352H-PE, Panasonic, Japan) for two days at 19 °C. Injected embryos developed on malt food. Each surviving female adult was mated with an uninfected male from line nu_0 to generate infected fly lines and each mating pair was kept separately in the mating cage. Three stably infected lines were generated, that is, ni_3, ni_6, and ni_8. To check for Wolbachia infection, five D. nigrosparsa females per line were randomly killed in each generation, and DNA was extracted and checked for infection by PCR using wsp81F and 691R primers.
4
Indentation of Mature Neuronal Cells
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F11s on their fifth and sixth differentiation days were used for experiments, with passages ranging from 14 to 23. Neuronal maturation was confirmed by immunohistochemistry with NeuN marker (Abcam, ab177487). Healthy F11s with sufficiently large round somas were selected for the study. While high cell density and well-connected neurons are desired for high spontaneous spiking rates, relative isolation of the cell is required to minimise the mechanical influence of neighbouring cells during indentation. As a compromise, cells whose somas were not in direct contact with those of other neurons, but still connected to other cells through their neurites, were chosen for the experiments.
Pipettes with resistances between 2 and 5 MΩ, as measured in the bath solution, were pulled from borosilicate glass capillaries (BF100-78-10, Sutter Instruments) through a 2.5 × 2.5 mm 2 box filament with a P-1000 puller (Sutter Instruments), following optimised parameters from Ref. [22] (link). Compositions of intra-and extracellular solutions can be found in Section 7. The liquid junction potential between the solutions was experimentally determined (to -11.9 mV), and the membrane potential values were corrected accord-
Pipettes with resistances between 2 and 5 MΩ, as measured in the bath solution, were pulled from borosilicate glass capillaries (BF100-78-10, Sutter Instruments) through a 2.5 × 2.5 mm 2 box filament with a P-1000 puller (Sutter Instruments), following optimised parameters from Ref. [22] (link). Compositions of intra-and extracellular solutions can be found in Section 7. The liquid junction potential between the solutions was experimentally determined (to -11.9 mV), and the membrane potential values were corrected accord-
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