Recovery of adherent cells after in situ electroporation monitored electrically.
Here we describe various experiments that address the efficiency of loading extracellular probes into the cytoplasm of adherent mammalian cells (normal rat kidney, Madin-Darby canine kidney, and African green monkey) by means of in situ electroporation. Subsequent cell recovery from the electroporation pulse was monitored electrically in real time for each condition. In this study, small, gold-film electrodes (5 x 10(-4) cm2) are used as culture substrates and at the same time as an electrode for both the application of the electroporating voltage pulse and the noninvasive electrical monitoring of cell recovery, using a technique referred to as ECIS. Electroporation has been performed by using ac sinusoidal voltage pulses of varying frequency, amplitude, and duration. Permeabilization and re-closure of the plasma membrane were evaluated by the uptake of the fluorescence probe, Lucifer Yellow, from the extracellularfluid. With the experimental setup described here, efficient electroporation was achieved with voltages less than 5 V. Using ECIS, we followed the morphological response of the cells to the electricfield-induced membrane permeabilization. For optimized electroporation conditions, cell recovery was completed in less than 1 h. The introduction of membrane-impermeable substances by electroporation and in situ monitoring of the cellular response mayfind many applications in cell biology.
Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Joachim
J
Wegener
Charles R
CR
Keese
Ivar
I
Giaever
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D002463:Cell Membrane Permeability; D002522:Chlorocebus aethiops; D004285:Dogs; D004566:Electrodes; D018274:Electroporation / Q000295:instrumentation* / Q000379:methods*; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D006046:Gold; D007546:Isoquinolines / Q000493:pharmacokinetics; D007668:Kidney / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D008856:Microscopy, Fluorescence; D051381:Rats; D012680:Sensitivity and Specificity; D013045:Species Specificity
D007546:Isoquinolines; D006046:Gold; C017475:lucifer yellow