Before recording, CM spheroids were stained with the Ca2+ indicator Fluo-4 AM. Fluo-4 AM (10 μM) (Thermo Fisher, catalog no. F14217) was prepared in 1× PBS and incubated at 37°C for 30 min. After incubation, the spheroids were washed three times with Tyrode’s solution prewarmed at 37°C. Each chip was glued to a printed circuit board (PCB) with soldered 36-pin connector (Omnetics, catalog no. A79024-001). The electrodes on the chip were wire-bonded to the Cu pads on the PCB using a manual wedge wire bonder (7476D Wire Bonder, West Bond). The chip was loaded onto the microscope stage, and the temperature inside the culture chamber was maintained by constant perfusion of Tyrode’s solution maintained at 37°C using an inline heater (ThermoClamp, Automate Scientific). The spheroids were encapsulated by 3D-SR-BAs. After initial set of recordings, the spheroids were treated with 20 μM blebbistatin and additional recording sets were obtained.

For electrical recordings, the Omnetics connector on the PCB was connected to a 32-channel amplifier (Intan Technologies, RHD2132), and the electrical signals were recorded using the Intan acquisition system (Intan Technologies, RHD2000) at an acquisition rate of 20 kHz. The optical recordings were performed using an upright microscope equipped with a resonant confocal scanner (Nikon A1R) under 20×/0.50 NA water immersion objective. All the recordings were performed in a grounded Faraday cage.

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