Synchronizing PC12 Cells for Dopamine Release

PC12 cells differentiated to neuronal-like phenotype in collagen IV-treated 12-well plates, were first synchronized all to the same circadian phase and cell cycle by a serum shock (50% HS and 50% DMEM HiGlutaXL) for 2 h at 37 °C [49 (link)] to achieve a synchronous release of dopamine [50 (link)]. Synchronization medium was then aspirated off, cells were washed with 3 × 1 mL and equilibrated for 15 min at 37 °C in 1 mL of Krebs Ringer HEPES (KRH) physiological buffer, following the recipe of Mount et al. [40 (link)] (25 mM HEPES/Tris, pH 7.4, 1.2 mM KH₂PO₄, 125 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO₄, 2.2 mM CaCl₂, 5.6 mM glucose, 1.0 mM ascorbic acid, 10 µM pargyline, 1.0 µM nomifensine). Basal or tonic pre-stimulation dopamine release was harvested, followed by the phasic release evoked by 60 mM K⁺ or eugenol in 1 mL KRH buffer, which is finally followed by the post-stimulation phase in 1 mL KRH for 5 min at 37 °C, which quantifies dopamine accumulation after the stimulation. All the three steps of dopamine release were collected sequentially during a time course of stimulation for 5, 15, 30, 60 and 120 min with 25 µM eugenol and for 5 min of stimulation with increasing concentrations of eugenol (1, 2, 5, 10 and 25 µM) together with 60 mM K⁺, as a positive control for functional dopamine release in PC12 cells. To remove cell debris, all samples of 1 mL incubation KRH at the end of treatments were centrifuged at 1000× g for 20 min at 4 °C and stored at −20 °C until dopamine levels were determined by ELISA assay.