Differentiation and Characterization of 1C11 Serotonin Neurons

1C11^5-HT cell culture was performed as described previously(82 (link)). Briefly, undifferentiated 1C11 cells were kept on 100 mm plates (Sarstedt) in DMEM Glutamax with 10% fetal bovine serum, 1% non-essential amino acids, 1% penicillin/streptomycin, and 1% L-glutamine (all media components by Life Technologies) at 37^°C and 5% CO₂. For differentiation to serotonin neuron-like cells (1C11^5-HT) 10.000 cells were transferred to 8-well imaging slides (Ibidi). Then, culture medium was supplemented with 1 mM dibutyryl cAMP and 0.05% cyclohexanecarboxylic acid for 4 days (media supplements by Sigma Aldrich). Treatment conditions: For determination of SERT cell surface density, medium was supplemented with 15 mM KCl and/or 1 μM escitalopram. For ASP⁺ live cell imaging, cells were transferred to FSCV buffer containing 50 μM ASP and/or 1 μM escitalopram and/or serotonin (0.1 or 1 μM). Electrical stimulation was applied after perfusing the imaging slides with ASP⁺-containing FSCV buffer. Dye-free buffer was applied 1 min after electrical stimulation before image acquisition.