Cellular response to high pulse repetition rate nanosecond pulses varies with fluorescent marker identity

Nanosecond electric pulses (nsEP's) are a well-studied phenomena in biophysics that cause substantial alterations to cellular membrane dynamics, internal biochemistry, and cytoskeletal structure, and induce apoptotic and necrotic cell death. While several studies have attempted to measure the effects of multiple nanosecond pulses, the effect of pulse repetition rate (PRR) has received little attention, especially at frequencies greater than 100 Hz. In this study, uptake of Propidium Iodide, FM 1–43, and YO-PRO-1 fluorescent dyes in CHO-K1 cells was monitored across a wide range of PRRs (5 Hz–500 KHz) using a laser-scanning confocal microscope in order to better understand how high frequency repetition rates impact induced biophysical changes. We show that frequency trends depend on the identity of the dye under study, which could implicate transmembrane protein channels in the uptake response due to their chemical selectivity. Finally, YO-PRO-1 fluorescence was monitored in the presence of Gadolinium (Gd³⁺), Ruthenium Red, and in calcium-free solution to elucidate a mechanism for its unique frequency trend. - Highlights: • Pulse repetition rate (PRR) is understudied in nanosecond electric pulsing. • 200 V pulses were applied to CHO-K1 cells from 5 Hz to 500 KHz. • Pulsing was repeated using a variety of fluorophores and imaging conditions. • The response is highly dependent on the fluorophore and the imaging conditions. • This may implicate protein channels in the nanoporation response.