2.3. Optical membrane potential monitoring with Di-8-ANEPPS

To load Di-8-ANEPPS into cell plasma membrane, coverslips with cells were incubated in the extracellular solution with 20 μM of the dye for 45 min at 4 °C. The coverslips were transferred into the glass-bottomed perfusion chamber and the excess dye was rinsed off.

The MP was measured by ratiometric imaging, which enabled a more reliable calibration, better signal-to-noise ratio, and reduced the impact of bleaching. The dye was excited alternately in 5-ms windows at 440 and 530 nm using fast wavelength switcher Lambda DG4 (Sutter Instruments, Novato, CA). We utilized a U-{"type":"entrez-nucleotide","attrs":{"text":"N71006","term_id":"1227586","term_text":"N71006"}}N71006 Di-8-ANEPPS filter set (Chroma Technology, Bellows Falls, VT) and a PlanApo N 60×/1.42 objective (Olympus). Emission was measured at 605 nm with an iXon Ultra 897 back-illuminated CCD Camera (Andor Technology, Belfast, UK). Dye emission ratio was calibrated against the membrane potential in voltage-clamped NG108 cells.

MP measurements with the dye typically began 2 s prior to psEP delivery and continued for 10 s after the exposure, at 100 image pairs/s. The image acquisition and on-line data analysis were accomplished with Metafluor v.7.5. (Molecular Devices). An FFT filter utility of Origin 8.0 (OriginLab Corporation, Northampton, MA) was employed for offline noise reduction.