To capture neural activity and synapse formation, both types of cells in the microfluidic devices were rinsed three times with PBS (without Ca2+ and Mg2+), recording medium [20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), 115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, and 13.8 mM glucose] was added with Fluo-8 AM (5 μM, 488 nm), and the mixture was incubated for 1 hour at 37°C. After loading Fluo-8 AM, the loading medium was replaced with the recording medium. Time-lapse movies were acquired for 10 min (resolution, 680 × 512 pixels; exposure time, 10 ms) using a fluorescent microscope (Axiovert 200, Zeiss).

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