Dmem glutamax

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.

Mice aged P0–P3 were euthanized by decapitation, and brains were dissected into PBS on ice. Brains of 6–8 mice were pooled, centrifuged at 500 × g for 10 min at 4 °C and resuspended in 0.25% Trypsin-EDTA (Thermo Fisher Scientific) at 37 °C for 10 min. DNase I (Thermo Fisher Scientific) was added at 1 mg/ml to the solution, and the brains were digested for 10 more minutes at 37 °C. Trypsin was neutralized by adding DMEM + GlutaMAX (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo Fisher Scientific), and cells were passed through a 70 µm cell strainer. Cells were centrifuged at 500 × g for 10 min at 4 C, resuspended in DMEM + GlutaMAX with 10% FBS 1% penicillin/streptomycin and cultured in T-75 flasks (Sarstedt), pre-coated with 2 µg/ml Poly-L Lysine (PLL, Provitro) at 37 °C in a humidified incubator with 5% CO₂ for 5–7 days until confluence was reached. Mixed glial cells were shaken for 30 min at 180 rpm, the supernatant was collected and the medium was changed, and then cells were shaken for at least 2 h at 220 rpm and the supernatant was collected and the medium was changed again. CD11b+ microglia were isolated from the collected supernatant using the CD11b Microbead Isolation kit (Miltenyi) according to the manufacturer’s instruction and seeded onto PLL-coated plates.

1C11 cells were cultured according to standard protocol.¹⁵, ¹⁶ Briefly, 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 Life Technologies) at 37°C and 5% CO₂. For differentiation to serotonergic 1C11 cells (1C11^5‐HT), 40,000 cells were transferred to a 3.5 cm plate (Sarstedt) containing 3 coverslips or slide chambers (Ibidi) for live cell imaging. Then, culture medium was supplemented with 1 mM dibutyryl cyclic adenosine monophosphate (cAMP) and 0.05% cyclohexanecarboxylic acid for 4 days. On day 4, 1C11^5‐HT were shown to display a complete 5‐HT metabolism.¹⁶ Fetal bovine serum in the medium, which contains 5‐HT, does not impact differentiation to a complete 5‐HT phenotype, but reduces 5‐HT synthesis, content and uptake by activation of 5‐HT_2B and 5‐HT_1B/D autoreceptors.¹⁶ As we did not quantify 5‐HT synthesis, content or uptake, this did not impact on our experiments. Additionally, medium was removed, and cells were washed three times in 1x phosphate‐buffered saline (PBS) prior to all experiments to remove medium residues and avoid any bias caused by 5‐HT in the medium. For each treatment, a randomized three‐digit code was applied before image acquisition. Image acquisition and analysis were performed under blinded conditions.