Feb 16, 2024

Public workspaceMeasuring dopamine release in human-derived iPSCs using High-Performance Liquid Chromatography (HPLC) coupled to Electrochemical Detection (ECD)

DOI
dx.doi.org/10.17504/protocols.io.q26g7p2bkgwz/v1
  • 1Oxford Parkinson’s Disease Center and Department of Physiology, Anatomy & Genetics, University of Oxford, Dorothy Crowfoot Hodgkin Building, South Parks Road, Oxford, OX1 3QU, UK;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
  • 3Kavli Institute for Nanoscience Discovery, University of Oxford, Dorothy Crowfoot Hodgkin Building, South Parks Road, Oxford, OX1 3QU, UK
Cláudia C. Mendes
Open access
DOI: dx.doi.org/10.17504/protocols.io.q26g7p2bkgwz/v1
Protocol Citation: Kaitlyn ML Cramb, Richard Wade-Martins, Stephanie J Cragg 2024. Measuring dopamine release in human-derived iPSCs using High-Performance Liquid Chromatography (HPLC) coupled to Electrochemical Detection (ECD). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7p2bkgwz/v1
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
We use this protocol and it's working
Created: February 12, 2024
Last Modified: February 16, 2024
Protocol Integer ID: 95107
Keywords: Dopamine release, HPLC-ECD, human iPSCs
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020370
Abstract
This protocol allows for the detection of evoked or tonic dopamine release from human iPSC-derived dopamine neurons using High-Performance Liquid Chromatography (HPLC) coupled to electrochemical detection.
Guidelines
This assay can be performed on any mature iPSC-derived dopamine neurons (DANs), but success has been particularly found on iPSC-DANs that have been grown in culture for 50-90 days following a modified Krik’s protocol. Optimal density found to be plated at 500 000 / well in a 24 well cell culture dish. Collected samples can be stored long-term at -80oC.

All steps are performed at Room Temperature (RT).
Materials
Reagents:

Equipment:
  • 4.6 x 150 mm Microsorb C18 reverse-phase column
  • Decade II ECD
  • Electrode (Antec Layden)

Making Ringers Buffer for evoked DA release:
Add each reagent to 500 mL dH2O
  • 1.2 mM CaCl2
  • 148 mM NaCl
  • 0.85 mM MgCl2
  • 40 mM KCl
  • pH 7.4, 300 osm
Making Ringers Buffer for tonic DA release:
Add each reagent to 500 mL dH2O
  • 1.2 mM CaCl2
  • 148 mM NaCl
  • 0.85 mM MgCl2
  • 2.7 mM KCl
  • pH 7.4, 300 osm

Making Mobile Phase Solution:
  • 13% HPLC grade methanol
  • 0.12 M Sodium phosphate monobasic dihydrate (NaH2PO4)
  • 0.8 mM Ethylenediaminetetraacetic acid (EDTA)
  • 0.5 mM 1-Octanesulphonic acid sodium salt (OSA)
  • pH 4.6

Preparation
Preparation
Add 1 µL of perchloric acid (PCA) to a labelled light-protected 1.5 mL Eppendorf tube for each sample.
Prepare Ringers Buffers for tonic or evoked release (see Materials). Store short-term at 4°C.
Dopamine (DA) Release Assay
Dopamine (DA) Release Assay
Aspirate media from hiPSC-DANs plated as stated in guidelines.
Wash hiPSC-DANs one time with 100 µL phosphate buffered saline (PBS).
Aspirate PBS from hiPSC-DANs.
Add 100 µL of Ringer’s Buffer for 5 minutes.
Collect Ringer’s Buffer in pre-labelled tubes containing PCA.
Snap freeze on dry ice (optional).
Note
Samples can be stored at -80oC long-term or immediately processed for HPLC.

HPLC-ECD
HPLC-ECD
If snap-frozen and stored at -80oC, thaw samples on ice.
Spin samples at 10 000 g for 10 minutes.
Run samples on HPLC column using a mobile phase running at 1 mL/min on a 4.6 x 150 mm Microsorb C18 reverse-phase column and Decade II ECD with a glassy carbon working electrode (Antec Layden) set at 0.7 V with respect to an Ag/AgCl reference electrode.
Calculate concentrate of dopamine released in sample compared to known standards.