Monitoring Electroporation-Induced Changes in Action Potential Generation in Genetically Engineered Tet-On Spiking HEK cells

Summary

For studying responses of excitable cells in vitro, the protocol describes optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells as well as changes in transmembrane voltage with automated extraction of relevant parameters.

Abstract

Excitable cells such as neuronal and muscle cells can be primary targets in rapidly emerging electroporation-based treatments. However, they can be affected by electric pulses even in therapies where they are not the primary targets, and this can cause adverse side effects. Therefore, to optimize the electroporation-based treatments of excitable and non-excitable tissues, there is a need to study the effects of electric pulses on excitable cells, their ion channels, and excitability in vitro. For this purpose, a protocol was developed for optical monitoring of changes in action potential generation due to electroporation on a simple excitable cell model of genetically engineered tet-on spiking HEK cells. With the use of a fluorescent potentiometric dye, the changes in transmembrane voltage were monitored under a fluorescence microscope, and relevant parameters of cell responses were extracted automatically with a MATLAB application. This way, the excitable cell responses to different electric pulses and the interplay between excitation and electroporation could be efficiently evaluated.

Introduction

In electroporation, high-voltage electric pulses cause an increase in plasma membrane permeability for molecules that are otherwise poorly permeant¹. Electroporation-based techniques are nowadays widely used in applications in medicine², biotechnology³, and food technology⁴.

Excitable cells such as neuronal and muscle cells are among the primary targets in rapidly emerging electroporation-based treatments in the heart, brain, and skeletal muscles⁵. Moreover, they can be affected by electric pulses even in therapies wh....

Protocol

1. Preparation of reagents and cell culture

1. Prepare the cell culture medium by supplementing Dulbecco's Modified Eagle's Medium-high glucose with 10% fetal bovine serum, 2 mM glutamine, penicillin-streptomycin (penicillin 100 U/mL, streptomycin 100 µg/mL), 2 µg/mL puromycin, 5 µg/mL blasticidin, and 200 µg/ mL geneticin.
2. Prepare phosphate-buffered saline (PBS) solution with 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH_2<.......

Discussion

S-HEK cells are a simple excitable cell model with well-defined sodium and potassium channels, Na_V1.5 and K_ir2.1⁹. This enables us to perform in-depth studies of how excitable cells respond to applied electric pulses and link the experimental data with mathematical models based on the well-known theoretical characteristics of the channels expressed in S-HEK cells. In our previous study⁸, we described the responses of these cells to 100 µs pulses o.......

Materials

Name	Company	Catalog Number	Comments
1.5 mL tube	Ratiolab	5615000
2-well Nunc Lab-Tek II Chambered #1.5 German Coverglass System	Thermo Fisher Scientific	155379
ADP305 Differential voltage probe	Teledyne LeCroy
AP015 Current probe	Teledyne LeCroy
Blasticidin	Thermo Fisher Scientific	A1113903
CO2 incubator	PHCbi	MCO -230AICUV
Doxycycline	Sigma-Aldrich/Merck	D9891
Dulbecco’s Modifed Eagle’s Medium high glucose growth medium	Sigma-Aldrich/Merck	D5671
ElectroFluor630 (Di-4-ANEQ(F)PTEA)	Potentiometric Probes	31795
Fetal bovine serum	Sigma-Aldrich/Merck	F2442
Geneticin	Thermo Fisher Scientific	10131035
Glutamine	Sigma-Aldrich/Merck	G7513
Laminar flow hood	Iskra Pio	MC 15-2
MATLAB	Mathworks
Penicillin–streptomycin	Sigma-Aldrich/Merck	P07681
Poly-L-lysine hydrobromide	Sigma-Aldrich/Merck	P9155-5MG
Pulse generator	Custom made		see reference11
Puromycin	Thermo Fisher Scientific	A1113803
T25 25cm² Tissue culture flask	TPP	90026
Tet-on spiking HEK cells	American Type Culture Collection ATCC	CRL-3479
Thunder Imager Live Cell system for fluorescence microscopy	Leica Microsystems
Trypsin-EDTA solution	Sigma-Aldrich/Merck	T3924
WavePro 7300A Oscilloscope	Teledyne LeCroy