Pipette puller

To isolate I_Kir4.1 from spinal astrocytes, whole-cell patch-clamp measurements were performed as described elsewhere [11 (link),25 (link),26 (link)]. Briefly, micropipettes were pulled from filament-containing borosilicate glass (1.5 mm OD × 0.86 mm ID × 100 mm L, Bio-Medical Instruments, Zöllnitz, Germany) with a pipette puller (Sutter Instruments, Novato, CA, USA) to have a resistance of 3–7 MΩ. The internal solution (pipette solution) consists of: 125 mM K-Gluconate, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM EGTA, 10 HEPES, 0.5 mM Na-GTP, and 2 mM Na₂ATP, pH with KOH (pH 7.2) and the external solution (bath solution) consists of: 14.4 mM NaHCO₃, 5.9 mM KCl, 2. 5 mM MgSO₄, 120.9 mM NaCl, 1.2 mM NaH₂PO₄, 2.5 mM CaCl₂, and 11.5 mM glucose (pH 7.4). The patch-clamp measurement was performed at a defined voltage, and spinal astrocytes were clamped at −70 mV with a depolarization range from −180 mV to 40 mV (in increments of 20 mV) in order to monitor the current-voltage relation curve (IV) of I_Kir4.1. The membrane potential, current density, and cell capacitance were additionally recorded.
Patch-clamp protocols were evaluated using the EPC10 amplifier, Patchmaster software (HEKA, Reutlingen, Germany) and Microsoft Excel. The Patchmaster software corrected “leakage currents” using an integrated P/4 protocol.

Isolated cardiomyocytes were investigated under a whole-cell patch clamp to record Ca²⁺ currents. Simply, the pipette puller (Sutter Instruments, Novato, CA, USA) was used to pull a glass pipette with a resistance of 3–5 MΩ filled with (in mM) 20 TEACL, 10 HEPES, 10 EGTA, 5 Mg-ATP, and 120 CsCl with a pH of 7.2 adjusted with CsOH. Myocardial cells were bathed in extracellular solution containing the following compounds (mM): 10 Glucose, 10 HEPES, 1.8 CaCl₂, 2 MgCl₂ and 140 TEACL with a pH of 7.4 adjusted with CsOH. This solution allows to reduce the interference from other currents. The electrodes were gently pressed onto the cell surface. The cell was aspirated and formed a high resistance seal at a zero potential difference between the inside and outside of the electrode. Axon patch 200B Amplifier (Axon Instruments, Union City, CA, USA) was filtered at 2 kHz and recorded the Ca²⁺ currents. Analyses of the data were carried out using the pCLAMP 10.7 software (Protocol: duration at a frequency of 10 Hz; the pulse wave form was 10 mV, 1 ms).

Membrane potential was measured with the ruptured whole-cell patch clamp. Microelectrodes were pulled from glass capillary (World Precision Instruments, Sarasota, FL, USA) by a pipette puller (Sutter Instrument, Novato, CA, USA) and polished with a micro forge (NARISHIGE, Japan). The microelectrodes used in experiments were typically 3–6 MΩ after they were filled with the internal solution. Both microelectrodes and solutions were prepared freshly before experiments. Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) was used to amplify the signal detected by the microelectrode. pClamp 10.4 software (Molecular Devices) was used for recording signals. Action potential (AP) data were analyzed with Cardiac Action Potential Analysis Software (CAPA) Package Distributed by Science Consulting Cardiac Cellular Electrophysiology UG (Essen, Germany) [23 ].

Whole-cell patch clamp recordings were performed in ventricular myocytes to measure the L-type Ca²⁺ currents. The pipette puller (Sutter Instrument, Novato, CA, United States) was employed for pulling the borosilicate glass microelectrodes of 3–5 MΩ resistance, which were later filled with a pipetted internal solution. I_Ca-L was recorded using an Axon Patch 200B amplifier with a 2 kHz applied to recordings. Data analyses were done using the Pclamp10.0 software (Axon Instruments, Union City, CA, United States). All experiments were performed in an air-conditioned room (23–25°C).