The calcium assay was performed using the Fluo-4 Direct Calcium Assay Kit according to the manufacturer’s protocol (F10471, Invitrogen). Briefly, cells at ~80% confluency were incubated with a 0.4× reagent at 37°C and 5% CO2 for 45 min and then at room temperature for 15 min. Subsequently, cells were trypsinized and seeded into the devices for imaging. A 0.4× reagent was included in the medium during the experiments. Intracellular calcium imaging in transfected HEK293 cells was carried out in cells loaded with 4.5 μM Fura2-AM (Fura-2-acetoxymethyl ester) (Invitrogen) as previously described (28).

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