Measuring dopamine release in human-derived iPSCs using High-Performance Liquid Chromatography (HPLC) coupled to Electrochemical Detection (ECD)
Stephanie J Cragg, Richard Wade-Martins, Kaitlyn ML Cramb
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.
Steps
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
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).
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 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.
