EF-induced cell migration experiments were performed as described previously [34 (link)]. Briefly, direct current was applied through agar-salt bridges via Ag/AgCl electrodes in Steinberg’s solution (consisting of 58 mM NaCl, 0.67 mM KCl and 0.44 mM Ca(NO3)2, 1.3 mM MgSO4 and 4.6 mM Tris base, pH 7.4) to pooled medium on either side of the galvanotaxis chamber. Cells were exposed to 0–100 mV/mm direct current EF for 1 h. Cell behavior was observed with a Carl Zeiss Observer Z1 inverted microscope with a Photometrics QuantEM EMCCD camera (Photometrics Inc., Tucson, AZ, USA) and MetaMorph NX software (Molecular Devices, Sunnyvale, CA, USA), and serial time-lapse images were captured. Cell migration was analyzed to determine directedness (cos θ) and track speed by using ImageJ software (NIH, Bethesda, MA, USA) with MTrackJ and Chemotaxis tool plugins. Briefly, trajectories of cells were pooled to make composite graphs. The directedness of migration was assessed as cos θ, where θ is the angle between the EF vector and a straight line connecting start and end positions of a cell. A cell moving directly to cathode would have a directedness of 1; a cell moving directly to the anode would have a directedness of −1. A value close to 0 represents random cell movement. Speed is the total length travelled by the cell divided by time.