Axopatch 200a patch clamp amplifier

Frozen aliquots of iPSC-derived neurons were thawed and plated on Biocoat glass cover slips (BD Biosciences) at a density of 10,000 cells per coverslip and maintained in a humidified incubator containing 5% CO₂ at 37°C. Whole-cell voltage-clamp recordings were carried out 6–10 days after plating. During recordings, cells were continuously perfused in HBTS at room temperature. Cells were voltage-clamped in the whole-cell configuration (at a holding potential of -60 mV) with an AxoPatch 200A patch-clamp amplifier (Molecular Devices, Sunnyvale, CA, USA). Pipettes were pulled from borosilicate glass (Type GC150TF-10, Harvard Apparatus, Kent, UK) using a commercial puller (Model P-87, Sutter Instruments, Novato, CA, USA) and had resistances between 2 and 6 MΩ when filled with a pipette solution containing (mM): 1 MgCl₂, 4 MgATP, 0.5 EGTA, 10 HEPES, 140 K gluconate (pH adjusted to 7.3 with KOH). Currents were recorded at 10 kHz using a DA/AD interface (Digidata 1322A, Molecular Devices, Sunnyvale, CA, USA). Drugs were applied using a multichannel perfusion system (Model BPS-8, Scientifica, Uckfield, UK) positioned 150 μm away from the recorded cell and controlled by Clampex 9 software (Molecular Devices, Sunnyvale, CA, USA).

Whole-cell voltage clamp recordings were made from cones. The recording chamber containing the isolated retina was mounted on a Nikon Eclipse E2000FN microscope (Nikon, Japan) and viewed with a Nikon 60× water immersion objective with infrared differential interference contrast and a video camera (Philips, Netherlands). Recording electrodes were pulled from borosilicate glass (BF-150-110-10; Sutter Instruments, Novato, CA, USA) with a Flaming/Brown micropipette puller (Model P-1000; Sutter Instruments, Novato, CA, USA). The impedances ranged from 8 MΩ to 12 MΩ when filled with pipette medium and measured in bathing solution. Pipettes were connected to an Axopatch 200A patch clamp amplifier (Molecular Devices, Sunnyvale, CA; four-pole low-pass Bessel filter setting, 2 kHz). Signal software (v. 3.07; Cambridge Electronic Design (CED), Cambridge, UK) was used to generate voltage command outputs and to acquire data. Signal software (v. 3.07; CED), MatLab (v2016b, MathWorks), Igor.pro (WaveMetrics, Portland, OR, USA) and Origin Pro (v8, Origin Lab Corporation), were used to analyze the data. All data shown are corrected for the junction potential.