Sep 16, 2020

Public workspaceHigh-Performance Liquid Chromatography (HPLC)

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. High-Performance Liquid Chromatography (HPLC). protocols.io https://protocols.io/view/high-performance-liquid-chromatography-hplc-9zdh726
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 02, 2019
Last Modified: September 16, 2020
Protocol Integer ID: 30469
Keywords: ND1014, N1, ND27760, ipsc, SNCA, Atoh2, Ngn2, HPLC
Abstract
This protocol describes High-Performance Liquid Chromatography (HPLC) for lines ND1014, N1, and ND27760 from Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons.
Materials
MATERIALS
ReagentPotassium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
Reagent Perchloric acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #244252
ReagentSavant SPD121P SpeedVac KitThermo ScientificCatalog #SPD121P
ReagentSavant Refrigerated Vapor TrapsThermo ScientificCatalog #RVT5105
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.
HPLC Preparation
HPLC Preparation
Replace neuron culture medium with Hanks’ balanced saline solution buffer with the addition of Concentration56 millimolar (mM) KCl .
Incubate for Duration00:15:00 at Temperature37 °C .

Incubation
Collect media and centrifuge to clear cell debris. Collect neuron pellet.
Centrifigation
Freeze immediately and store at Temperature-80 °C .
Pause
HPLC Analysis
HPLC Analysis
Thaw samples.
Concentrate using a vacuum (Savant SPD 121P) connected with a refrigerated vapor trap (Savant RVT 5105).
Resuspend freeze-dried samples in Concentration10 millimolar (mM) perchloric acid .

Analyze monoamines using HPLC electrochemical detection by dual channel Coulochem III electrochemical detector (model 5300).
Analyze
Separate monoamines using a reverse phase C18 column (3-mm 3 150-mm C-18 RP-column) with a flow rate of 0.600 ml/minute.
Quantify monoamine concentrations by comparison of the area under the curve to known standard dilutions.
Computational step