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

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.

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).

If snap-frozen and stored at -80^oC, 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.