title: Cellular response to high pulse repetition rate nanosecond pulses varies with fluorescent marker identity.
abstract: 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 (Gd(3+)), Ruthenium Red, and in calcium-free solution to elucidate a mechanism for its unique frequency trend.
authors.affiliation: Duke University, Department of Biomedical Engineering, 101 Science Drive, Durham, NC, USA. Electronic address: zachary.steelman@duke.edu.
authors.forename: Zachary A
authors.initials: ZA
authors.lastname: Steelman
authors.affiliation: General Dynamics Information Technology, Fort Sam Houston, TX, USA.
authors.forename: Gleb P
authors.initials: GP
authors.lastname: Tolstykh
authors.affiliation: Radio Frequency Bioeffects Branch, Air Force Research Laboratories, Ft. Sam Houston, San Antonio, TX, USA.
authors.forename: Hope T
authors.initials: HT
authors.lastname: Beier
authors.affiliation: Radio Frequency Bioeffects Branch, Air Force Research Laboratories, Ft. Sam Houston, San Antonio, TX, USA.
authors.forename: Bennett L
authors.initials: BL
authors.lastname: Ibey
mesh_terms: D000818:Animals; D001583:Benzoxazoles / Q000378:metabolism; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000378:metabolism*; D005682:Gadolinium / Q000378:metabolism; D006801:Humans; D053758:Nanoparticles / Q000737:chemistry*; D011419:Propidium / Q000378:metabolism; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D011806:Quinolinium Compounds / Q000378:metabolism; D012430:Ruthenium Red / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D001583:Benzoxazoles; C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D011806:Quinolinium Compounds; D012430:Ruthenium Red; C089813:YO-PRO 1; D011419:Propidium; D005682:Gadolinium; D002118:Calcium
keywords: CHO-K1; Confocal microscopy; Fluorescence microscopy; Nanosecond pulse; nsEP