Sep 17, 2020

Public workspaceElectrophysiological Recordings

This protocol is a draft, published without a DOI.
  • Yingchao Xue1,2,
  • Xiping Zhan3,
  • Shisheng Sun4,
  • Senthilkumar S. Karuppagounder5,6,7,
  • Shuli Xia2,5,
  • Valina L Dawson5,6,7,8,9,
  • Ted M Dawson5,6,7,8,10,
  • John Laterra2,5,8,11,
  • Jianmin Zhang1,
  • Mingyao Ying2,5
  • 1Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology;
  • 2Hugo W. Moser Research Institute at Kennedy Krieger;
  • 3Department of Physiology and Biophysics, Howard University;
  • 4College of Life Sciences, Northwest University;
  • 5Department of Neurology, Johns Hopkins University School of Medicine;
  • 6Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine;
  • 7Adrienne Helis Malvin Medical Research Foundation;
  • 8Department of Neuroscience, Johns Hopkins University School of Medicine;
  • 9Department of Physiology, Johns Hopkins University School of Medicine;
  • 10Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine;
  • 11Department of Oncology, Johns Hopkins University School of Medicine
  • Neurodegeneration Method Development Community
    Tech. support email: ndcn-help@chanzuckerberg.com
Anita Broellochs
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External link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344911/
Protocol Citation: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying 2020. Electrophysiological Recordings. protocols.io https://protocols.io/view/electrophysiological-recordings-9vbh62n
Manuscript citation:
Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. Xue Y, Zhan X, Sun S, Karuppagounder SS, Xia S, Dawson VL, Dawson TM, Laterra J, Zhang J, Ying M. Stem Cells Transl Med. 2019 Feb;8(2):112-123. doi: 10.1002/sctm.18-0036. Epub 2018 Nov 1. PMID: 30387318
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
These protocols were published in: Xue Y, Zhan X, Sun S, et al. Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. Stem Cells Transl Med. 2019;8(2):112–123. doi:10.1002/sctm.18-0036
Created: November 28, 2019
Last Modified: September 17, 2020
Protocol Integer ID: 30339
Keywords: ND1014, N1, ND27760, ipsc, SNCA, Atoh2, Ngn2
Abstract
This protocol explains Quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR) of lines ND1014, N1, and ND27760 from Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons.
Safety warnings
Please refer to the Safety Data Sheets (SDS) for safety and environmental hazards.
Before start
Obtain approval to work with human stem cells from an appropriate Institutional Review Board.
Voltage-Clamp Recording
Voltage-Clamp Recording
Perform voltage-clamp recordings at Temperature35 °C in a chamber perfused with regular artificial cerebrospinal fluid flowing at 3 ml/minute.


Regular artificial cerebrospinal fluid:
  • Concentration124 millimolar (mM) NaCl
  • Concentration2.5 millimolar (mM) KCl
  • Concentration1.3 millimolar (mM) MgCl2
  • Concentration2.5 millimolar (mM) CaCl2
  • Concentration1 millimolar (mM) NaH2PO4
  • Concentration26.2 millimolar (mM) NaHCO3
  • Concentration20 millimolar (mM) glucose at Ph7.4 , equilibrated with 95% O2 and 5% CO2, ∼310 mosm)
Note
Patch electrodes should be pulled from borosilicate glass and have resistances of 2.0–4.0 MΩ when filled with an intracellular solution.

Intracellular solution:
  • Concentration135 millimolar (mM) KMeSO4
  • Concentration5 millimolar (mM) KCl
  • Concentration5 millimolar (mM) HEPES ,
  • Concentration0.25 millimolar (mM) EGTA‐free acid
  • Concentration2 millimolar (mM) Mg‐ATP
  • Concentration0.5 millimolar (mM) GTP
  • Concentration10 millimolar (mM) phosphocreatine‐tris at Ph7.3 , ∼290 mosm



Identify neurons using a ×10 objective mounted on an upright microscope with transmitted light.
Imaging
Visualize their neuronal somata through a ×40 water immersion objective using IR differential interference contrast optics.
Cell somatic recordings should be made using an Axopatch 200B amplifier in combination with pClamp 10.7 software (Molecular Devices).
Note
  • Voltage clamp at −70 mV.
  • Monitor Rseriesand Rinput using a 2.5‐mV 100‐ms depolarizing voltage step in each recording sweep.
  • Filter current traces at 5 kHz, digitize at 10 kHz using a Digidata 1440 interface, and store for off‐line analysis.
  • Monitor resting membrane potential and the action potential for more than 5 minutes before drug applications.


Add Tetraethylammonium (TEA) and ZD 7288 (Sigma‐Aldrich) the artificial cerebrospinal fluid, to block K+ orIhcurrents, respectively.