title: Multiple nanosecond electric pulses increase the number but not the size of long-lived nanopores in the cell membrane.
abstract: Exposure to intense, nanosecond-duration electric pulses (nsEP) opens small but long-lived pores in the plasma membrane. We quantified the cell uptake of two membrane integrity marker dyes, YO-PRO-1 (YP) and propidium (Pr) in order to test whether the pore size is affected by the number of nsEP. The fluorescence of the dyes was calibrated against their concentrations by confocal imaging of stained homogenates of the cells. The calibrations revealed a two-phase dependence of Pr emission on the concentration (with a slower rise at<4μM) and a linear dependence for YP. CHO cells were exposed to nsEP trains (1 to 100 pulses, 60ns, 13.2kV/cm, 10Hz) with Pr and YP in the medium, and the uptake of the dyes was monitored by time-lapse imaging for 3min. Even a single nsEP triggered a modest but detectable entry of both dyes, which increased linearly when more pulses were applied. The influx of Pr per pulse was constant and independent of the pulse number. The influx of YP per pulse was highest with 1- and 2-pulse exposures, decreasing to about twice the Pr level for trains from 5 to 100 pulses. The constant YP/Pr influx ratio for trains of 5 to 100 pulses suggests that increasing the number of pulses permeabilizes cells to a greater extent by increasing the pore number and not the pore diameter.
authors.affiliation: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA. Electronic address: 2andrei@pakhomov.net.
authors.forename: Andrei G
authors.initials: AG
authors.lastname: Pakhomov
authors.affiliation: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA.
authors.forename: Elena
authors.initials: E
authors.lastname: Gianulis
authors.affiliation: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA.
authors.forename: P Thomas
authors.initials: PT
authors.lastname: Vernier
authors.affiliation: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA.
authors.forename: Iurii
authors.initials: I
authors.lastname: Semenov
authors.affiliation: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA; Department of Electrical and Computer Engineering, Old Dominion University, Norfolk, VA, USA.
authors.forename: Shu
authors.initials: S
authors.lastname: Xiao
authors.affiliation: Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA.
authors.forename: Olga N
authors.initials: ON
authors.lastname: Pakhomova
mesh_terms: D000818:Animals; D001583:Benzoxazoles / Q000737:chemistry; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism* / Q000528:radiation effects; D002463:Cell Membrane Permeability / Q000502:physiology* / Q000528:radiation effects; D006224:Cricetinae; D003412:Cricetulus; D004558:Electric Stimulation / Q000379:methods*; D004574:Electromagnetic Fields; D018274:Electroporation / Q000379:methods*; D058608:Nanopores; D011419:Propidium / Q000737:chemistry; D011674:Pulse; D011806:Quinolinium Compounds / Q000737:chemistry; D059008:Time-Lapse Imaging
chemical_list: D001583:Benzoxazoles; D011806:Quinolinium Compounds; C089813:YO-PRO 1; D011419:Propidium
keywords: Electropermeabilization; Electroporation; Membrane permeability; Nanopores; Nanosecond pulses; nsEP