A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

Summary

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

Abstract

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.  Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

Introduction

The application of electrical current to a cell causes the formation of pores on the cell membrane, by a process termed electroporation. The pores allow the passage of a variety of nonpermeant molecules through the membrane. The electrical field can be controlled precisely, so that the pores formed are very small and reclose rapidly, with minimal disturbance to the cellular physiology. Interestingly, adherent cells can be grown on a glass slide coated with conductive and transparent indium-tin oxide (ITO) and electroporated in situ, that is on the surface where they grow, without being detached to be electroporated in suspension. Cells can grow very well on t....

Protocol

1. Plating the Cells in the Electroporation Chambers

1. In a laminar-flow hood, trypsinize the cells using sterile technique as usual.
   NOTE: It is very important to eliminate by centrifugation all traces of trypsin, because they may hinder spreading of the cells on the glass, hence the formation of adherens and gap junctions.
2. Pipette 1 ml of the cell suspension in the sterile electroporation chambers provided with the in situ electroporator (Figure 1), and place in a 37 ^oC, CO₂ incubator.
   NOTE: Cell adhesion can be improved by plating on fibronectin, collagen, poly-lysine or cell and tiss....

Results

Figure 2 shows rat liver epithelial T51B cells²², electroporated with LY and photographed under fluorescence (panel A and B), or phase-contrast (C) illumination, following fixation and washing. In panel A, the edge of the electroporated area is marked in red. The gradient of fluorescence to the right of the red line denotes transfer through gap junctions. In Figure 3A and 3B, the quantitation of gap junctional communication is shown: The cells at the edge of t.......

Discussion

Critical steps in the protocol

Electroporated material
The purity of the material to be electroporated is very important. If the cells are very flat, then higher concentrations of the tracking dye must be used (up to 20 mg/ml for LY) and in such cases, purity is even more important than in cells with a more spherical shape¹. Besides LY a large variety of other dyes or nonpermeant molecules have been employed, such as a series of Alexa dyes to use as probes for c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Cellgro http://www.cellgro.com/	50-013-PB
DMEM without Calcium	Hyclone (Thermo scientific: http://www.thermoscientific.com)	SH30319-01
Donor Calf Serum	PAA: http://www.paa.com/	Cat.# B15-008
Fetal Bovine Serum	PAA: http://www.paa.com/	Cat.# A15-751
Electroporation apparatus	Cell Projects Ltd UK: http://www.cellprojects.com/	ACE-100
Chambers	Cell Projects Ltd UK	ACE-04-CC	4-wells
Chambers	Cell Projects Ltd UK	ACE-08-CC	8-wells
Lucifer Yellow	Cell Projects Ltd UK	ACE-25-LY	High purity
CelTak	BD Biosciences	354240	Cell and tissue adhesive
Fibronectin	Sigma Aldrich	F1141
Collagen	BD Biosciences	354236
Poly-Lysine	Sigma Aldrich	P8920