Axopatch 200b patch clamp amplifier

iPSC-dSN were seeded at 3.95 x 10⁴ cells/cm² on 5 x 5 mm plastic coverslips coated with matrigel. Cells were cultured and maintained on coverslips for the first 48 hours followed by siRNA pool delivery for 72 hours and subsequently treated with either vehicle or paclitaxel for 48 hours, as described above. Whole-cell patch-clamp recordings were conducted as previously reported [26 (link), 27 (link)] in voltage-clamp mode at room temperature (∼22°C) using Axopatch 200B patch-clamp amplifier (Molecular Devices). Data were acquired on a Windows-based Pentium IV computer using the pClamp (8.0) software (Molecular Devices). Fire-polished electrodes (1.5–2.5 MΩ) were fabricated from borosilicate glass capillaries (Sutter Instrument Company) using a P-97 puller (Sutter Instrument Company). The standard electrode solution consisted of 140 mm CsF, 10 mm NaCl, 1.1 mm EGTA, and 10 mm HEPES, pH 7.3. The standard extracellular bathing solution contained 130 mm NaCl, 30 mm TEA chloride, 1 mm MgCl2, 3 mm KCl, 1 mm CaCl2, 0.05 mm CdCl2, 10 mm HEPES, and 10 mm D-glucose, pH 7.3.

Proteoliposomes were thawed and dispensed in 0.5–1 μL drops on a 35 mm glass-bottom dish. The drops were dried in a vacuum chamber in the dark overnight. Proteoliposomes were rehydrated with 20 μL buffer (5  mM HEPES pH 7.2, 200  mM KCl). Each cake was firstly covered with a buffer drop and then let surface tension connect drops. Rehydrating proteoliposomes were placed within a humid chamber at 4°C before patching. Recordings were made at room temperature using Clampex v.10.7 data acquisition software (as part of the pClamp v.10.7 suite) with an Axopatch 200B Patch Clamp amplifier and Digidata 1550B digitizer (Molecular Devices) at a bandwidth of 1  kHz and digitized at 500  kHz. A pressure clamp (ALA Scientific) was used to form seals. Pipette solution was 10 mM HEPES pH 7.2, 150 mM KCl, 3 mM MgCl₂, and 5 mM EGTA. Bath solution was 10 mM HEPES pH 7.3, 135 mM NaCl, 15 mM KCl, 1 mM CaCl₂, and 3 mM MgCl₂. Borosilicate glass pipettes were pulled and polished to a resistance of 2–5  MΩ when filled with pipette solution.

Whole-cell voltage-clamp recordings were obtained from the target astrocytes or neurons at room temperature using an Axopatch 200B patch-clamp amplifier and pClamp 11.0.3 software (Molecular Devices, SanJose, CA) as described previously (Mohrmann et al., 1999 (link)). The patch pipette was filled with intracellular solution containing (in mM): 110 KCl, 20 HEPES, 10 EGTA, 0.25 CaCl2 with pH adjusted to 7.3. The standard extracellular solution contained (in mM) 130 NaCl, 3 KCl, 2.5 CaCl₂, 1 MgCl₂, and 20 HEPES, with pH adjusted to 7.3. The high [K⁺] extracellular solution contained (in mM) 112 NaCl, 40 KCl, 2.5 CaCl₂, 1 MgCl₂, and 20 HEPES, with pH adjusted to 7.3. AMPA receptor-mediated miniature EPSCs (1 μM TTX, 10μM gabazine added) were recorded at a holding potential of -60 mV.

Shortly before the experiments, the cells (i.e., GH₃ cells and mHippoE-14 neurons) were dissociated, and an aliquot of cell suspension was transferred to a home-made recording chamber that was mounted on the stage of a DM-IL inverted microscope (Leica, Wetzlar, Germany), and then left to settle. The cells were immersed at room temperature (20–25 °C) in HEPES-buffered normal Tyrode’s solution, the composition of which is described above. For the recordings, patch electrodes were fabricated from borosilicate glass capillaries (#34500; Kimble Products, Vineland, NJ, USA) on a Narishige PP-830 puller (Narishige, Tokyo, Japan), then fire-polished with an MF-83 microforge (Narishige). The resistances existing between the standard pipette and the bathing solution ranged between 3 and 5 MΩ. Recordings of different types of ionic currents were measured in the whole-cell mode using the standard patch-clamp technique with either an RK-400 (Bio-Logic, Claix, France) or an Axopatch 200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA, USA) [32 (link)], which was interfaced via a Digidata 1440A to a PC running pCLAMP suite software (Molecular Devices). The liquid junction potentials were corrected shortly before seal formation was established.

Recordings were obtained in the cell-attached patch configuration^{59 (link)} at a temperature of 21 °C, essentially as described previously^{58 (link)}. Patch pipettes were pulled from glass capillary tubes (No.7052, King Precision Glass) and coated with Sylgard (Dow Corning). The bath solution contained (in mM): 142 KCl, 5.4 NaCl, 1.8 CaCl₂, 1.7 MgCl₂, and 10 HEPES, with the pH adjusted to 7.4 by addition of KOH. The pipette solution contained (in mM): 142 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.7 MgCl₂, and 10 HEPES, with the pH adjusted to 7.4 by addition of NaOH. Specified concentrations of acetylcholine chloride, with or without ethanol, were added to the pipette solution. Before establishing a cell-attached patch, the pipette offset potential was manually zeroed, and following formation of a giga-ohm seal, a defined membrane potential was established via a command voltage applied to the interior of the patch pipette. Single channel currents were recorded using an Axopatch 200B patch clamp amplifier (Molecular Devices) with the gain set at 100 mV/pA and the internal Bessel filter at 100 kHz. The current output was sampled at intervals of 2 μs using a National Instruments model BNC-2090 A/D converter with a PCI 6111e acquisition card, and recorded to the hard disk of a PC computer using the program Acquire (Bruxton).