Sep 17, 2020

Public workspace6-Hydroxydopamine (6-OHDA) Treatment and Neurite Tracing in miDA Neurons

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. 6-Hydroxydopamine (6-OHDA) Treatment and Neurite Tracing in miDA Neurons. protocols.io https://protocols.io/view/6-hydroxydopamine-6-ohda-treatment-and-neurite-tra-9zfh73n
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: December 03, 2019
Last Modified: September 17, 2020
Protocol Integer ID: 30471
Keywords: ND1014, N1, ND27760, ipsc, SNCA, Atoh2, Ngn2, 6-Hydroxydopamine (6-OHDA), Neurite Tracing, miDA, Neurons
Abstract
This protocol describes 6-Hydroxydopamine (6-OHDA) Treatment and Neurite Tracing in miDA Neurons from Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons.
Materials
MATERIALS
ReagentCalcein, AM, cell-permeant dyeThermo FisherCatalog #C3100MP
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.
Cell Seeding
Cell Seeding
Plate miDA neurons (3 × 103 per well) in a 384-well plate coated with poly-D-lysine and laminin.
Calcein AM Assay
Calcein AM Assay
Prepare fresh 6-OHDA in vehicle solution (0.15% ascorbic acid in H2O).
Note
Control cells should be treated with vehicle solution alone.


Incubate cells with fresh 6-OHDA solution for Duration24:00:00 .

Incubation
Stain live cells with Concentration1 micromolar (µM) calcein-AM .
Note
The calcein AM assay is based on the conversion of the cell permeant nonfluorescent calcein AM dye to the fluorescent calcein dye by intracellular esterase activity in live cells.

Image using a confocal microscope.
Imaging
Calcein-AM Analysis
Calcein-AM Analysis
Quantify neurite length using high content analysis software (HCA-Vision V2.2.0. CSIRO).
Analyze