To detect serotonin release into medium during neuronal differentiation, we collected NDM for the culture every week. To detect activity-dependent serotonin release, Krebs’ Ringer’s solution with specified K+ concentration was used as previously described42. Briefly, cultured cells were washed four times with Krebs’ Ringer’s solution containing 130 mM NaCl, 3 mM KCl, 2 mM CaCl2, 0.8 mM MgSO4, 10 mM glucose and 20 mM HEPES (pH 7.4) before the medium was collected with depolarizing Krebs’ Ringer’s solution containing 83 mM NaCl, 50 mM KCl, 2 mM CaCl2, 0.8 mM MgSO4, 10 mM glucose and 20 mM HEPES (pH 7.4). To detect drug- induced serotonin release, tramadol (Sigma) and escitalopram oxalate (EO, from Tocris Bioscience) were applied to the culture at different dosages and timing, and the NDM was collected at specific time points. The collected media were stored in −80 °C before detection.
