Dopamine Quantification in Neuron Supernatants

The supernatant was collected from neurons differentiated on top of PA6 and kept directly at −80°C until the time of analysis. Before their analysis, the medium samples were previously deproteinized with 50 μl of homogenization medium (100 ml miliQ H2O, 100 mg of sodium metabisulphite (Sigma), 10 mg of EDTA‐Na (Sigma), 100 mg of cysteine (Sigma) and 3.5 ml of HClO₄ concentrated (Scharlau, 70%)); centrifuged at 3,723 g for 30 min at 4°C, and the supernatant was filtered (0.45 μm, Millipore) for a posterior HPLC injection. The concentration of 3,4‐Dihydroxy‐L‐phenylalanine (L‐Dopa), dopamine (DA), and 3,4‐dihydroxyphenylacetic acid (DOPAC) in supernatant samples was determined using an HPLC system with a Waters 717 plus autosampler (Waters Cromatografia), a Waters 515 pump, a 5 μm particle size C18 column (100 × 46 mm, Kinetex EVO, Phenomenex), and a Waters 2465 amperometric detector set at an oxidation potential of 0.75 V. The mobile phase consisted of 0.15 M NaH₂PO₄.H₂O, 0.57 mM 1‐octane sulfonic acid, 0.5 mM EDTA (pH 2.8, adjusted with phosphoric acid), and 7.4% methanol and was pumped at 0.9 ml/min. The total sample analysis time was of 50 min and the L‐Dopa, DA, and DOPAC retention times were 2.06, 3.94, and 4.25 min respectively. The detection limit was of 2–3 fmol (injection volume 60 μl). The corresponding content of the DA metabolite was normalized to the protein concentration previously determined by Bradford method detection.