To characterize the electrophysiological properties of the iCMs, an MEA recording system (Multichannel Systems, Reutlingen, Germany) was used. The MEAs have 60 microelectrodes, with a diameter of 30 µm, positioned on an 8 × 8 grid with 200-µm spacing. Freshly isolated neonatal FBs (fibroblasts) were plated on gelatin-coated MEA culture plates and were transduced with MGT retrovirus in the presence of shRNA targeting Becn1 or nontargeting (NT) control. The reprogramming was performed as aforementioned. Standard measurements of field potential were performed at 5 kHz in culture medium at 37°C (76 (link)). Data acquisition and analysis were finished using MC_Rack software. The number of spikes recorded from electrodes showing extracellular electrograms was calculated in a time frame of 30 s.
Mea recording system
Manufactured by MultiSciences Biotech
Sourced in Germany
The MEA recording system is a specialized lab equipment used to measure and record the electrical activity of cells, particularly neurons, in a multi-electrode array (MEA) setup. The system allows for the simultaneous recording of electrical signals from multiple electrodes, providing a powerful tool for the study of neural network dynamics and function.
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5 protocols using mea recording system
1
Electrophysiological Characterization of iCMs
2
Characterizing Electrophysiology of iPSC-Derived Cardiomyocytes
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A multi-electrode array (MEA) recording system (Multichannel Systems, Reutlingen, Germany) was used to characterize the electrophysiological properties of human iPSC-derived cardiomyocytes. The spontaneously beating EBs were plated on fibronectin-coated MEA plates and incubated at 37 °C. The extracellular electrograms were recorded in DMEM/HEPES (D5796; Sigma-Aldrich) at 37 °C, and the obtained data were subsequently analyzed with MC_Rack (Multichannel Systems). The extracellular electrograms were used to determine field potential duration (FPD) and detect arrhythmic events. FPD was defined as the time interval between the initial deflection of the field potential and its return to baseline. FPD measurements were normalized (cFPD) to the rate using Bazett's correction formula: cFPD=FPD/(RR interval)1/2.
3
Evaluating Cardiomyocyte Electrophysiology via MEA
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The electrophysiological properties of the cardiomyocytes derived from iPS cells were evaluated using a microelectrode array (MEA) recording system (Multichannel Systems, Reutlingen, Germany). Beating cell clusters at day 10 post-plating were transferred onto MEA plates coated with 0.1% gelatin and 10 µg/mL fibronectin. Responsiveness to pharmacological agents was determined 4–6 days later at 37 °C in Krebs-Ringer buffer (composition in mM: 125 NaCl, 5 KCl, 1 Na2HPO4, 1 MgSO4, 20 HEPES, 5.5 glucose, 2 CaCl2; pH 7.4). The responsiveness of cells to isoproterenol hydrochloride (1–1000 nM, Sigma-Aldrich) and carbamylcholine (1–1000 nM, Sigma-Aldrich) was tested. Each cell cluster was treated with all drugs in random order and cells were allowed to recover to their baseline contraction in fresh Krebs-Ringer buffer in between drug treatments. Extracellular field potentials were recorded at baseline and 2 min after addition of drugs. Data were analysed offline with MC Rack version 4.3.5 software for beating rate, RR interval and extracellular field potential duration (FPD) as previously described35 (link),36 (link). FPD measurements were normalized (corrected FPD, cFPD) with the Bazzet correction formula: cFPD = FPD/√(RR interval).
4
Electrophysiological Characterization of iPSC-Derived Cardiomyocytes
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Randomly chosen iPSCs from two healthy volunteers (control) and the patient with LQTS3/BrS were used for electrophysiological characterization. Two independent iPSC lines were used from the patient. The electrophysiological properties of beating EBs were assessed using a multielectrode array (MEA) recording system (Multichannel Systems, Reutlingen, Germany). The beating EBs were transferred to fibronectin-coated MEA plates. The extracellular potential was recorded in DMEM/HEPES (D5796; Sigma-Aldrich) at 37 °C at 10 kHz temporal resolution. The electrograms recorded were used to determine field potential duration (FPD). This parameter corresponds to the action potential duration (APD) and reflects the QT interval. The estimated FPD was defined as the time interval between the initial deflection of the field potential and the time of the maximum of the next wave. FPD measurements were corrected (cFPD) for the rate of beating of the EBs using Fridericia’s formula: cFPD = FPD/3√(RR interval).
5
Extracellular Field Potential Recording of Beating Cardiomyocytes
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Extracellular field potential recording of the beating colonies was performed using the microelectrode array (MEA) recording system (Multichannel Systems, Reutlingen, Germany). Beating EBs at day 10 postplating were transferred onto MEA plates precoated with 0.1% gelatin and 10 μg/mL fibronectin. Responsiveness to isoproterenol hydrochloride (1–100 nM, Sigma-Aldrich) and carbamylcholine (1–100 nM, Sigma-Aldrich) was determined 4–6 days later at 37°C in Krebs-Ringer buffer (composition in mM: 125 NaCl, 5 KCl, 1 Na2HPO4, 1 MgSO4, 20 HEPES, 5.5 glucose, and 2 CaCl2; pH 7.4). Each cell cluster was treated with all drugs in random order, and cells were allowed to recover to their baseline contraction in fresh Krebs-Ringer buffer in between drug treatments. Extracellular field potentials were recorded at baseline and 2 minutes after the addition of drugs. Data were analyzed offline with MC Rack version 4.3.5 software for the beating rate, RR interval, and extracellular field potential duration (FPD) as previously described [7 (link), 44 (link), 46 (link)]. FPD measurements were normalized (corrected FPD, cFPD) with the Bazzet correction formula: cFPD = FPD/√(RR interval).
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