High Performance Liquid Chromatography (HPLC) and sample preparation

Prior to harvesting mDA neurons, all cells were incubated in phenol-free medium for 24 hours with and without the presence of 80micromolar (µM) L-Dopa (Sigma).

For extracellular metabolites, media after 24 hours was mixed 1:1 in 0.8Molarity (M) ice cold perchloric acid.

Samples were incubated on ice for 0h 10m 0s , followed by centrifugation at 12000x g,4°C .

The supernatant was removed and frozen in dry ice for HPLC analysis.

For intracellular metabolites, after 24 hours, the cells were removed and pelleted by centrifugation at 1200x g,4°C.

The cells were washed once in PBS and lysed on ice using lysis buffer (10mM Tris (pH 7.4), 1mM EDTA, 320mM sucrose in HPLC grade water).

Lysate was mixed with 1:1 in 0.8Molarity (M) ice cold perchloric acid and incubated on ice for 0h 10m 0s.

The samples were centrifuged at 12000x g,4°C and the supernatant was harvested and frozen for HPLC analysis.

Quantification of neurometabolites (DOPAC, 3-OMD, 5-HIAA, HVA and dopamine) was carried out using reverse phase HPLC and an electrochemical detector

The mobile phase (flow rate 1.5ml/min) contained 16% methanol, 20mM sodium acetate trihydrate, 12.5mM citric acid monohydrate, 3.35mM 1-octanesulfonic acid, 0.1mM EDTA disodium and adjusted to pH 3.45 with 12 M hydrochloric acid (HCl).

The stationary phase was maintained at 27°C .

The detector electrode was set at 450mV and screening electrode at 50mV.

50µl of sample was injected and calculated against a 500nM external standard solution of the 5 compounds of interest made in HPLC grade water acidified with 12M HCl.

Peak areas were quantified with EZChrom Elite™ chromatography data system software, version 3.1.7 (JASCO UK Ltd., Great Dunmow, UK).