Ix3 zdc

6-well plates were coated with 50 µg/ml poly-L-lysine (Sigma-Aldrich) for 20 min and air-dried. Cells were plated and cultured to confluency before a 10 µl pipet tip was used to create a cross-shaped wound across the monolayer. Samples were imaged on a widefield microscope (IX-81, Olympus) equipped with an LED lamp (Excelitas Technologies), an Orca 16-bit charge-coupled device camera (Hamamatsu), an automated z-drift compensation IX3-ZDC (Olympus), an automated stage (Prior Scientific), an environmental chamber (Live Cell Instrument) and using a 10X objective (MPlanFL N 10X, 0.3 NA, Olympus). Cell motility was recorded at 10 min intervals over 48 h. Manual cell tracking was performed using the TrackMate plugin through Fiji^{53 (link)}. The track number, the spot coordinates and the frame number were exported. Computation of the velocity and persistence were done using a custom made Matlab code.

Fluorescence was excited with a 475 nm LED (Olympus) and monitored using an inverted microscope (IX83, Olympus) equipped with a ×150 1.45 NA oil-immersion objective. A continuous acquisition was performed at 50 ms/frame for 120 s using a cooled EMCCD camera (iXon life 888, ANDOR). The Z-drift compensation system (IX3-ZDC, Olympus) was used to ensure a constant position of the focal plane during imaging. All experiments were conducted at 37°C within a whole-microscope incubator chamber (TOKAI HIT). Field simulation was performed at 1 Hz precisely synchronized with the beginning of an acquisition frame using a pair of platinum electrodes and controlled by the software via Master-9 stimulus generator (A.M.P.I.). Samples were perfused with bath solution containing 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 15 mM glucose, 50 μM APV, 10 μM CNQX, adjusted to pH 7.4.