Epc 10 patch clamp amplifier

Electrical activity was measured from β cells within intact mouse islets using the perforated-patch technique. All measurements were performed between 32–34 °C. During the experiments, the islets were immobilized using a wide-bore glass suction pipette^{35 (link)} and superfused with a modified Krebs–Ringer solution containing (mM) 140 NaCl, 3.6 KCl, 1.3 CaCl₂, 0.5 MgSO₄, 10 HEPES, 0.5 NaH₂PO4, and 5 NaHCO₃ at pH 7.4 with NaOH and glucose as indicated. The solution within the pipette contained 76 K₂SO₄, 10 KCl, 10 NaCl, 1 MgCl₂, and 5 HEPES (in mM, pH 7.35 using KOH). Membrane perforation was effected by inclusion of the pore-forming antibiotic amphotericin B (240 μg/ml) in the intracellular buffer. Islet cell types were established by functional fingerprinting³⁶ . Measurements were performed using an EPC-10 patch-clamp amplifier (HEKA Electronics, Ludwigshafen/Rhein, Germany) and PatchMaster software (version 2 × 91).

Using an EPC10 patch clamp amplifier (HEKA Electronics) controlled by Patchmaster (HEKA), whole-cell recordings were conducted 24–72 hours after splitting of RPE cells or transfection of HEK293 cells. 1.5 mm thin-walled glass with filament (WPI Instruments) were pulled and fashioned to micropipettes, and filled with internal solution containing (in mM): 130 CsCl, 10 EGTA, 1 MgCl₂, 2 MgATP (added fresh), 10 HEPES (pH 7.4, adjusted by CsOH), and CaCl₂ to obtain the desired free Ca²⁺ concentration (maxchelator.stanford.edu/CaMgATPEGTA-TS.htm). Series resistance was usually 1.5–2.5 MΩ. There was no electronic series resistance compensation. External solution contained (in mM): 140 NaCl, 15 glucose, 5 KCl, 2 CaCl₂, 1 MgCl₂ and 10 HEPES (pH 7.4, adjusted by NaOH). Solution osmolarity was between 310 and 315. A family of step potentials (−100 to + 100 mV from a holding potential of 0 mV) were used to generate I-V curves. Currents were sampled at 25 kHz and filtered at 5 or 10 kHz. Traces were acquired at a repetition interval of 4 s^{48 (link)}. All experiments in this study were carried out at ambient temperature (23 ± 2 °C).

Electrophysiological analyses were conducted 72–96 h after transfection. Whole-cell patch clamp recording was performed with an EPC10 patch clamp amplifier (HEKA Electronics) controlled by Patchmaster v2x90.5 (HEKA). Micropipettes were pulled and fashioned from filamented 1.5 mm thin-walled glass (WPI Instruments), and filled with internal solution containing (in mM): 130 CsCl, 1 MgCl₂, 10 EGTA, 2 MgATP (added fresh), 10 HEPES (pH 7.4, adjusted by CsOH). The desired no Ca²⁺ and 1 μM free Ca²⁺ concentrations were obtained by adding 0 and 8.7 mM CaCl₂, respectively (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaMgATPEGTA-TS.htm). Series resistance was typically 1.5–2.5 MΩ, with no electronic series resistance compensation. The recipe of external solution was (in mM): 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES (pH 7.4, adjusted by NaOH) and 15 glucose. Solution osmolarity was 290–310 mOsm/L with glucose. Traces were acquired at a repetition interval of 4 s^{23 (link)}. Currents were sampled at 25 kHz and filtered at 5 or 10 kHz. I–V curves were generated from a group of step potentials (−100 to +100 mV from a holding potential of 0 mV). Experiments were conducted at room temperature (23 ± 2 °C).

An EPC10 patch clamp amplifier (HEKA Electronics) controlled by Patchmaster (HEKA) was utilized to conduct whole-cell recordings 24–72 hr after splitting of RPE cells or transfection of HEK293 cells. Micropipettes were pulled and fashioned from 1.5 mm thin-walled glass with filament (WPI Instruments) and filled with internal solution containing (in mM): 130 CsCl, 10 EGTA, 1 MgCl₂, 2 MgATP (added fresh), 10 HEPES (pH 7.4, adjusted by CsOH), and CaCl₂ to obtain the desired free Ca²⁺ concentration (maxchelator.stanford.edu/CaMgATPEGTA-TS.htm). Series resistance was usually 1.5–2.5 MΩ. No electronic series resistance compensation was used. External solution contained (in mM): 140 NaCl, 15 glucose, 5 KCl, 2 CaCl₂, 1 MgCl₂, and 10 HEPES (pH 7.4, adjusted by NaOH). Solution osmolarity was between 310 and 315. A family of step potentials (−100 to +100 mV from a holding potential of 0 mV) were used to generate I-V curves. Currents were sampled at 25 kHz and filtered at 5 or 10 kHz. Traces were acquired at a repetition interval of 4 s (Yang et al., 2014b (link)). All experiments in this study were carried out at ambient temperature (23 ± 2°C).

Electrophysiological analyses were conducted 48–72 hours after transfection. Whole-cell patch-clamp recording was performed with an EPC10 patch-clamp amplifier (HEKA Electronics) controlled by Patchmaster (HEKA). Micropipettes were pulled and fashioned from filamented 1.5 mm thin-walled glass (WPI Instruments) and filled with internal solution containing 130 mM CsCl, 1 mM MgCl₂, 10 mM EGTA, 2 mM MgATP (added fresh), and 10 mM HEPES (pH 7.4, adjusted by CsOH). The desired Ca²⁺ concentrations were obtained by adding CaCl² (Ca/Mg/ATP/EGTA Calculator v1 was used to calculate CaCl2, https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaMgATPEGTA-TS.htm). Series resistance was typically 1.5–2.5 MΩ, with no electronic series resistance compensation. The recipe of external solution was 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES (pH 7.4, adjusted by NaOH), and 15 mM glucose. Solution osmolarity was 290~310 mOsm/L with glucose. Traces were acquired at a repetition interval of 4 seconds. Currents were sampled at 25 kHz and filtered at 5 or 10 kHz. I-V curves were generated from a group of step potentials (–100 to +100 mV from a holding potential of 0 mV). Experiments were conducted at room temperature (23°C ± 2°C). Whole-cell patch-clamp data were processed off-line in Patchmaster.