Quantification of electroporative uptake kinetics and electric field heterogeneity effects in cells.
We have conducted experiments quantitatively investigating electroporative uptake kinetics of a fluorescent plasma membrane integrity indicator, propidium iodide (PI), in HL60 human leukemia cells resulting from exposure to 40 mus pulsed electric fields (PEFs). These experiments were possible through the use of calibrated, real-time fluorescence microscopy and the development of a microcuvette: a specialized device designed for exposing cell cultures to intense PEFs while carrying out real-time microscopy. A finite-element electrostatic simulation was carried out to assess the degree of electric field heterogeneity between the microcuvette's electrodes allowing us to correlate trends in electroporative response to electric field distribution. Analysis of experimental data identified two distinctive electroporative uptake signatures: one characterized by low-level, decelerating uptake beginning immediately after PEF exposure and the other by high-level, accelerating fluorescence that is manifested sometimes hundreds of seconds after PEF exposure. The qualitative nature of these fluorescence signatures was used to isolate the conditions required to induce exclusively transient electroporation and to discuss electropore stability and persistence. A range of electric field strengths resulting in transient electroporation was identified for HL60s under our experimental conditions existing between 1.6 and 2 kV/cm. Quantitative analysis was used to determine that HL60s experiencing transient electroporation internalized between 50 and 125 million nucleic acid-bound PI molecules per cell. Finally, we show that electric field heterogeneity may be used to elicit asymmetric electroporative PI uptake within cell cultures and within individual cells.
Kennedy
S M
SM
Department of Electrical and Computer Engineering, University of Wisconsin, Madison, Wisconsin 53706, USA. smkennedy@wisc.edu
Ji
Z
Z
Hedstrom
J C
JC
Booske
J H
JH
Hagness
S C
SC
D001704:Biopolymers / Q000493:pharmacokinetics*; D002462:Cell Membrane / Q000378:metabolism* / Q000528:radiation effects; D003198:Computer Simulation; D004574:Electromagnetic Fields; D018274:Electroporation / Q000379:methods*; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008657:Metabolic Clearance Rate / Q000528:radiation effects; D008856:Microscopy, Fluorescence / Q000379:methods*; D008954:Models, Biological
D001704:Biopolymers