2.3. DiBAC4(3) Membrane Potential Measurement

Cells were plated in a 384-well plate pre-treated with Cell-Tak (Corning, Fisher Scientific, Waltham, MA, USA) at 25,000 cells/well. Cells were then loaded with 250 nM DiBAC4(3) (Bis-(1,3-Dibutylbarbituric Acid) Trimethine Oxonol) (Thermo Fisher, Waltham, MA, USA) for 20 min in 37 °C in 5% tissue culture incubator. DiBAC4(3) was added to cells in Live Cell Imaging Solution and 50 mM KCl physiological saline solution (PSS) (as mentioned above). The indicator was removed, and cells were loaded with their respective buffer. Membrane potential fluorescence indicated by DiBAC4(3) was captured using excitation and emission of 488 and 510, respectively, using the BIOTEK Cytation 5 imager. Fluorescence was captured every 30 s and analyzed as mentioned previously.