Bottom glass coated petri dishes

Cortical tissue from dead, aborted human fetuses at 7–9 weeks post-conception was obtained from Lund and Malmö University Hospitals according to the guidelines approved by Lund/Malmö Ethical Committee. The tissue was micro-dissected under a stereomicroscope (Leica, Germany) and frozen in Stem Cell Banker (AMSBIO). At the moment of culture preparation, frozen tissue was resuspended in BrainPhys Media and dissociated mechanically by pipetting. Then, the cell suspension was diluted with BrainPhys supplemented with 1:50 B27 without retinoic acid and 1:100 penicillin-streptomycin and plated on bottom-glass-coated Petri dishes (Ibidi), reaching a final density of 200–400 neurons/mm². 1 week after plating (DD7), cells were infected with AAV-GCaMP6s (1 μL/mL).

Human iPSC-derived cortical NPCs from healthy male newborns (Axol Bioscience) were grown in Neuronal Expansion Media (Axol Bioscience), supplemented with 1:100 penicillin-streptomycin (Gibco) and 1:50 B27 (Gibco). For differentiation into cortical neurons, cells were detached with Unlock Solution (Axol Bioscience) and transferred to bottom-glass-coated Petri dishes (Ibidi) at a final density of 200–400 neurons/mm² (coating protocol detailed in supplemental information). This time point was set as DD0. Media was switched at DD1 to Neural Differentiation XF Media (Axol Bioscience) and finally at DD4 to BrainPhys (Stem Cell Technologies), supplemented with 1:50 B27 without retinoic acid (Gibco) and 1:100 penicillin-streptomycin, which was renewed every 3–4 days onward. 1 week after starting differentiation (DD7), cells were infected with the AAV containing the fluorescence indicator GCaMP6s under the control of Syn-I promoter (1 μL/mL).

For cultures of rat embryonic cells (E18–E19) from the cortex, all procedures were approved by the Animal Experimentation Ethics Committee (CEEA) of the University of Barcelona, under order DMAH-5461, in accordance with the regulations of the Generalitat de Catalunya (Spain). Briefly, dissection was carried out in ice-cold L-15 medium (Gibco), enriched with 0.6% glucose and 0.5% gentamicin (Sigma-Aldrich). The extracted brain cortices were isolated from the meninges, transferred to cell media, and dissociated mechanically by pipetting. The final suspension was plated on the bottom-glass-coated Petri dishes (Ibidi), corresponding to DD0. The density after seeding was 200–400 neurons/mm². 2 days after plating (DD2), cells were infected with AAV-GCaMP6s (1 μL/mL). At DD5, cultures were treated with 0.5% FUDR for 3 days to restrict glial proliferation. Afterward, cells were maintained with periodic medium renewal every 3 days, using BrainPhys, supplemented with 1:50 B27 without retinoic acid and 1:100 penicillin-streptomycin.