Dissecting Pathways; in situ Electroporation for the Study of Signal
Transduction and Gap Junctional
Electroporation has been used for the introduction
of DNA and proteins, as well as various nonpermeant
drugs and metabolites into cultured mammalian cells
(reviewed in Neumann et al.
, 2000; Chang et al.
Most electroporation techniques for adherent cells
involve the delivery of the electrical pulse while the
cells are in suspension. However, the detachment of
these cells from their substratum by trypsin or EDTA
can cause significant metabolic alterations (Matsumura et al.
, 1982), while the efficient incorporation of proteins,
peptides, or drugs without cellular damage is an especially
crucial requirement, since contrary to DNA, no
convenient large-scale method exists for the selection of
viable from damaged cells or cells where no introduction
took place after electroporation, for most proteins
or drugs of interest. For these reasons, a number of
approaches have been taken to bypass this problem
(Kwee et al.
, 1990; Yang et al.
This article describes a technique where cells are
grown on a glass surface coated with electrically conductive,
optically transparent indium-tin oxide (ITO)
at the time of pulse delivery. This coating promotes
excellent cell adhesion and growth, allows direct visualization
of the electroporated cells, and offers the
possibility of ready examination due to their extended
morphology. The procedures described are applicable
to a wide variety of nonpermeant molecules, such as
peptides (Giorgetti-Peraldi et al.
, 1997; Boccaccio et al.
1998; Bardelli et al.
, 1998), oligonucleotides (Boccaccio et al.
, 1998; Gambarotta et al.
, 1996), radioactive
nucleotides (Boussiotis et al.
, 1997), proteins
(Nakashima et al.
, 1999), DNA (Raptis and Firth, 1990),
or drugs (Marais et al.
, 1997). These compounds can be
introduced alone or in combination, at the same or different
times, in growth-arrested cells or cells at different
stages of their division cycle. After introduction
of the material, cells can be either extracted and
biochemically analysed or their morphology and biochemical
properties examined in situ
. In a modified
version, this assembly can be used for the study of
intercellular, junctional communication. The instant
introduction of the molecules into essentially 100% of
the cells makes this technique especially suitable for
kinetic studies of effector activation. Unlike other techniques
of cell permeabilization, under the appropriate
conditions, in situ
electroporation does not affect cell
morphology, the length of the G1 phase of serumstimulated
cells (Raptis and Firth, 1990), the activity of
the extracellular signal regulated kinase (Erk½ or
Erk), or two kinases commonly activated by a number
of stress-related stimuli, JNK/SAPK and p38hog
(Robinson and Cobb, 1997), presumably because the
pores reseal rapidly so that the cell interior is restored
to its original state (Brownell et al.
II. MATERIALS AND
The purity of the material to be electroporated is of
paramount importance. Substances such as detergents, preservatives, or antibiotics could kill the cells into
which they are electroporated, even if they have no
deleterious effects if added to the culture medium of
Dulbecco's modification of Eagle's medium
(DMEM) is from ICN (Cat. No. 10-331-22). Fetal calf
serum (Cat. No. 2406000AJ) and phosphate-buffered
saline (PBS, Cat. No. 20012-027) are from Gibco Life
Technologies. Calf serum is from ICN (Cat. No. 29-131-
54). EGF is from Intergen (Cat. No. 4110-80). HEPES
(Cat. No. H-9136), Lucifer yellow CH dilithium salt
(Cat. No. L-0259), trypsin (Cat. No. T-0646), MgCl2
(Cat. No. M-8266), Triton (Cat. No. T-6878), deoxycholate
(Cat. No. D-5760), EDTA (Cat. No. E-5134),
EGTA (Cat. No. E-3889), phenylmethylsulfonyl fluoride
(PMSF, P-7626), aprotinin (Cat. No. A-6279),
leupeptin (Cat. No. L-2023), benzamidine (Cat. No. B-
6506), dithiothreitol (DTT, Cat. No. D-9779), CaCl2
(Cat. No. C-4901), Tris (Cat. No. T-6791), paraformaldehyde
(Cat. No. P-6148), bovine serum albumin (BSA,
Cat. No. A-4503), SigmaFast DAB kit (Cat. No. D-9167
and Cat. No. U-5005), and sodium orthovanadate (Cat.
No. S-6508) are from Sigma. Extran-300 detergent (Cat.
No. B80002), SDS (Cat. No. 44244), NP-40 (Cat. No.
56009), glycerol (Cat. No. B10118), and peroxide (B
80017) are from BDH. CelTakTM
is from BD Biosciences
(Cat. No. 354240). The cell staining kit, including
goat serum, secondary antibody, and avidin-biotin
complex, was from Vector Labs (Vectastain kit Cat. No.
PK-6101). Rabbit antipeptide antibodies against the
double threonine and tyrosine phosphorylated (activated)
Erk½ kinase are from Biosource International
(Cat. No. 44-680). When stored frozen in aliquots, they
are stable for more than 5 years. They are used at
1:500 for immunostaining and 1:10,000 for Western
blotting. Three- and 6-cm tissue culture dishes are
from Corning or Sarstedt. Please note that only these
brands of plates fit the electroporation stand.
The Grb2-SH2 binding peptide is based on the
sequence flanking the Y1068
of the EGF receptor (PVPEPmp-
INQS, MW 1123). To enhance stability of the
phosphate group, the phosphotyrosine analog,
phosphono-methylphenylalanine (Pmp), which
cannot be cleaved by phosphotyrosine phosphatases
yet binds to SH2 domains with high affinity and specificity
(Otaka et al.
, 1994), is incorporated at the position
of phosphotyrosine. The Pmp monomer is custom synthesized
by Color your enzyme Inc., (Kingston, Ontario,
Canada). As control, we used the same peptide containing
phenylalanine at the position of Pmp. Peptides
are synthesized by the Queen's University Core Facility
using standard Fmoc chemistry.
The system for electroporation in situ
Model EZ-16) can be purchased from Ask Science
Products Inc. (Kingston, Ontario, Canada, phone: 613
545-3794). The inverted, phase-contrast and fluorescence
microscope, equipped with filters for Lucifer
yellow and fluorescein, was from Olympus (Model
The technique of in situ
electroporation can be used
equally effectively for large-scale biochemical experiments
(Giorgetti-Peraldi et al.
, 1997; Boccaccio et al.
1998; Bardelli et al.
, 1998) or for the detection of biochemical
or morphological changes in situ
(Raptis et al.
2000a). Cells are grown on glass slides coated with
conductive and transparent indium-tin oxide (ITO).
The cell growth area is defined by a "window" formed
with an electrically insulating frame made of Teflon as
shown. A stainless-steel electrode is placed on top of
the cells resting on the frame and an electrical pulse of
the appropriate strength is applied, as illustrated in
Fig. 1 (see also chapter 43 by Raptis et al.
). The technique
can be applied to a large variety of adherent cell
types (Brownell et al.
, 1996). Cells that do not adhere
well can be grown and electroporated on the same conductive
slides coated with CelTakTM
used according to the manufacturer's instructions.
A. Electroporation of Peptides into Large
Numbers of Cells for Large-Scale Biochemical
Experiments. Use of Fully Conductive Slides
|FIGURE 1 Electroporation electrode and slide assembly. Cells
are grown on glass slides coated with conductive and transparent
ITO within a "window" cut into a Teflon frame as shown. The
window can be different sizes, depending on the cell growth area
required. The peptide solution is added to the cells and introduced
by an electrical pulse delivered through the electrode set, which is
placed directly on the frame. Dotted lines point to the positions of
negative and positive electrodes during the pulse. Three slide configurations
are described. (A) Partly conductive slide assembly, with
electroporated (a) and non-electroporated (c) cells growing on the
same type of ITO-coated surface. (b) area where the conductive
coating has been stripped, exposing the non-conductive glass underneath.
Cells growing in areas b and c are not electroporated (Fig. 3).
(B) Partly conductive slide assembly for use in the examination of
gap junctional, intercellular communication. Arrow points to the
transition line between conductive and non-conductive areas (Fig.
4). (C) Fully conductive slide assembly for use in biochemical experiments.
In the setup shown, cell growth area can be up to 7 × 15 mm,
but larger slides and electrodes offer larger areas, up to 32 × 10mm
To study the effect of protein interactions in vivo
cellular functions, such complexes can be disrupted
through the introduction of peptides corresponding to
the proteins' point(s) of contact. An example of this
approach is described here.
Growth factors such as the epidermal growth factor
(EGF) stimulate cell proliferation by binding to, and
activating, membrane receptors with cytoplasmic tyrosine
kinase domains. In vitro
binding and receptor
mutagenesis studies have shown that ligand engagement
induces receptor autophosphorylation at distinct
tyrosine residues, which constitute docking sites for a
number of effector molecules, such as the growth
factor receptor-binding protein 2 (Grb2), which are
recruited to specific receptors through modules
termed Src-homology 2 (SH2) domains (reviewed in Schlessinger
, 2000). Grb2 binds to the receptors for
PDGF and EGF at a number of sites, an event activating
the Sos/Ras/Raf/Erk pathway, which is central to
the mitogenic response stimulated by many growth
factors. Previous results indicated that a synthetic phosphopeptide corresponding to the Grb2-binding
site of the EGF receptor (EGFR, flanking the EGFR
PVPE-pY-INQS), when made in tandem with
peptides that allow for translocation across the cell
membrane, could inhibit EGF-mediated mitogenesis
and Erk activation in newt myoblasts induced by
1 ng/ml EGF, but was less effective at 10ng/ml
(Williams et al.
, 1997). To better determine the functional
consequences of disrupting the association of Grb2 per se
with different receptors in vivo
cells, we delivered large quantities of this
peptide into intact, living NIH3T3 fibroblasts by in situ
: Grow cells to confluence in DMEM with
10% calf serum. Seven days postconfluence, collect
the culture supernatant and dilute 1:1 with fresh
DMEM. Growth-arrest cells by incubating in spent
medium prepared from the same line.
Lucifer yellow solution, 5mg/ml
: To make 10ml, dissolve
50mg Lucifer yellow in 10ml calcium-free DMEM.
Stable at 4°C for at least a month.
: The peptide concentration required varies
with the strength of the signal to be inhibited. For
the inhibition of the EGF-mediated Erk activation,
prepare a solution of 5-10mg/ml (~5-10mM
the Grb2-SH2-blocking peptide (PVPE-pmp-INQS,
MW 1123 Da) in calcium-free DMEM (see Comment
Epidermal growth factor
: To make a 10,000× stock solution,
dissolve 100 µg of lyophilised EGF in 100 µl
sterile water and freeze in 5µl aliquots. Just prior
to the experiment, add 1µl stock solution to 10ml
calcium-free DMEM (final concentration, 100ng/
ml). The stock solution is stable at -20° or -70°C for
up to 2 months.
HEPES, pH 7.4, 150mM
, 100mm NaF, 2mM
vanadate, 0.5 mM
PMSF, 10µg/ml aprotinin,
10µg/ml leupeptin, 1% Triton X-100.
- Choice of slides. For Western blotting experiments
on cell extracts following electroporation, use
fully conductive slides (Fig. 1C). Since the custommade
peptide is usually the most expensive reagent in
this application, to avoid waste, choose the smallest
possible cell growth area which provides a sufficient
number of cells. Cell growth areas of 32 × 10mm are
generally sufficient to detect Erk½ activity inhibition
by the Grb2-SH2 blocking peptide in EGF-stimulated,
mouse NIH3T3 fibroblasts. In this case, the volume
of the solution under the electrode is ~140µl and
will contain approximately 700-1,400µg peptide in
calcium-free DMEM. If fewer cells suffice, then slides
with a cell growth area of 7 × 15mm can be used,
requiring ~40µl of peptide solution. However, for the
determination of [3H]thymidine uptake, cell growth
areas of 7 × 4mm are preferred and they require only
~14µl of solution (see Comment 2).
- Plate the cells. Uniform spreading of the cells is
very important, as the optimal voltage depends in part on the degree of cell contact with the conductive
surface (see Comment 3). Add a sufficient amount of
medium (DMEM containing 10% calf serum) to cover
the slide (approximately 9 ml for a 6 cm dish). Pipette
the cell suspension in the window cut in the Teflon
frame (Fig. 1) and place the petris in a tissue-culture
incubator until confluent.
- Prior to the experiment, starve the cells overnight
in DMEM without serum. Alternatively, cells can be
incubated in spent medium for 48h; this treatment
offers wider margins of voltage tolerance (see Comment 3).
- Prior to pulse application, remove the growth
medium and wash the cells gently once with calciumfree
- Carefully wipe the Teflon frame with a folded
Kleenex tissue to create a dry area on which a meniscus
can form (see Pitfall 1).
- Add the peptide solution to the cells with a
micropipettor in calcium-free DMEM.
- Carefully place the electrode on top of the cells
and clamp it in place. To ensure electrical contact, a
sufficient amount of growth medium or PBS should be
present under the positive contact bar. Make sure there
are no air bubbles under the negative electrode. If necessary,
the electrode can be sterilized with 80% ethanol
before the pulse and the procedure carried out in a
laminar flow hood, using sterile solutions.
- Apply three to six pulses of the appropriate
voltage and capacitance (see Comment 3).
- Remove the electrode set. Since usually only a
small fraction of the material enters the cells, the
peptide solution may be carefully aspirated and used
again. However, care must be exercised so that the cells
do not dry (see Pitfall 1).
- Add serum-free growth medium and incubate
the cells for 2-5 min at 37°C to recover.
- Add EGF to the medium to a final concentration
of 100ng/ml for 5min. Controls receive the same
volume of calcium-free DMEM.
- Extract the cells with 50µl extraction buffer for
a cell growth area of 32 × 10mm. For smaller cell
growth areas, the voltage can be adjusted accordingly.
- To detect activated Erk½, load 100µg of total
cell extract protein on an acrylamide-SDS gel and
analyse by Western immunoblotting using the antibody
directed against the dually phosphorylated, i.e.,
activated, form of Erk½.
- For examination of the effect of the peptide upon
[3H]thymidine incorporation into DNA, serum-starve
50% confluent, NIH3T3 cells as described earlier and
electroporate in the presence or absence of peptide.
Incubate in medium with or without EGF for 12h at
37°C, followed by a 2h incubation with 50µCi/ml
[3H]thymidine. Wash the cells with PBS and measure
acid-precipitable counts. Growth areas of 4 × 7 mm are
sufficient for this experiment, and [3H]thymidine can
be added to the window only, in a volume of ~50µl which is held in place by surface tension.
As shown in Fig. 2A, electroporation of the Grb2-
SH2 blocking peptide caused a dramatic reduction in
EGF-mediated Erk activation in mouse NIH3T3 cells
at growth factor concentrations permitting full receptor
stimulation (compare lanes 2 and 3 with lane 4). In
addition, electroporation of this peptide reduced EGFmediated
H]thymidine uptake (Fig. 2B). In contrast,
the same peptide had only limited or no effect on Erk
activation triggered by HGF, although it could inhibit
PDGF signalling (Raptis et al.
, 2000a). These findings
demonstrate that the in situ
described can very effectively inhibit growth factorstimulated
mitogenesis and thereby detect the differential
specificity in the coupling of activated receptor
tyrosine kinases to the Erk cascade.
B. Electroporation for the Study of
Morphological Effects or Biochemical Changes in situ: Use of Partly Conductive Slides
|FIGURE 2 (A) The Grb2-SH2 blocking peptide inhibits EGF-mediated Erk activation in living cells;
detection by Western blotting. The Grb2-SH2 blocking peptide was electroporated into NIH3T3 cells
growing on fully conductive slides (inset, Fig. 1C, cell growth area 32 × 10mm) and growth-arrested by serum
starvation. After a 5min incubation in DMEM, cells were stimulated with 100 ng/ml EGF (lanes 2-4) for
5 min. Proteins in detergent cell lysates were resolved by polyacrylamide gel electrophoresis and analysed
by Western blotting using the antibody against the dually phosphorylated, active Erk enzymes. Lane 1,
control, unstimulated cells; lane 2, control non-electroporated, EGF-treated cells; lane 3, cells electroporated
with the control, phenylalanine-containing peptide and EGF stimulated; and lane 4, cells electroporated with
the Grb2-SH2-binding peptide and EGF stimulated. From Raptis et al. (2000), reprinted with permission.
(B) The Grb2-SH2 blocking peptide inhibits EGF-mediated DNA synthesis. The Grb2-SH2 blocking
peptide (Pmp) or its phenylalaline-containing counterpart (phe) were electroporated at the indicated concentrations
into NIH3T3 cells growing on fully conductive slides (Fig. 1C, cell growth area, 4 × 7mm) and
growth-arrested by serum starvation. Following incubation at 37°C and stimulation with EGF or 10% calf
serum for 12h, cells were labelled for 2h with 50µCi/ml [3H]thymidine and acid-precipitable radioactivity
was determined. Numbers represent the mean ± SE from three experiments. From Raptis et al., (2000),
reprinted with permission.
Assessment of Erk activity by Western blotting
following electroporation of the Grb2-SH2 blocking
peptide can reveal the involvement of this domain in
growth factor-mediated Erk activation. However, to
ensure that the treatment itself does not cause cell
stress, examination of cellular morphology, in conjunction
with measurement of gene product activity
by immunocytochemistry, offers a distinct advantage.
This approach can demonstrate the specificity of action
of the Grb2-SH2 binding peptide, as well as examine
the distribution of signal inhibition across the cell
layer. An added advantage is that it requires a small
number of cells, hence a substantially smaller volume
of the peptides (~14µl in the setup shown in Fig. 1A),
compared to Western blotting (~140µl for a cell growth
area of 32 × 10mm), which could be a significant consideration
given their production costs. To precisely
assess small background changes in morphology or
gene expression levels, the presence of non-electroporated
cells side by side with electroporated ones can
offer a valuable control and this can be achieved by
growing the cells on a conductive slide where part of
the coating has been stripped by etching with acids,
thus exposing the non-conductive glass underneath.
However, as shown in Fig. 3, area a
, the slight tinge
of the glass combined with the more effective staining
of cells growing on ITO (possibly due to a chemical
attraction of different reagents to the coating) can create problems in the interpretation of results. In
addition, it was found that a number of cell lines grow
slightly better on the conductive, ITO-coated glass
than the nonconductive area, possibly due to the fact
that the ITO-coated surface is not as smooth as glass,
thus providing a better anchorage for the growth of
adherent cells (Folkman and Moscona, 1978). As a
result, cell density may be higher on the conductive
than the etched side, which could have important
implications if cell growth effects are being studied. It
follows that, to assess the effect of the peptide, it is
important to compare the staining and morphology of
electroporated cells with non-electroporated ones
while both are growing on the same type of surface.
This was achieved by plating the cells on a slide where the conductive coating was removed in the pattern
shown in Fig. 1A (Firth et al.
, 1997). A thin line of plain
glass separates the electroporated and control areas
while etching extends to area Fig. 1A, d so that there is
no electrical contact between the positive contact bar
and area Fig. 1A,c. Application of the pulse results in
electroporation of the cells growing in area Fig. 1A, a
exclusively, while cells growing in area Fig. 1A,b or c
do not receive any pulse. In this configuration, electroporated
cells are being compared to nonelectroporated
ones, while both are growing on ITO-coated
glass. Because the coating is only ~1,600Å thick, this
transition zone does not alter the growth of cells across
it and is clearly visible microscopically, even under a
cell monolayer (Figs. 3 and 4).
|FIGURE 3 The Grb2-SH2 blocking peptide inhibits EGF-mediated Erk activation in living cells; detection
by immunocytochemistry. The Grb2-SH2 blocking peptide (A and B) or its control, phenylalaninecontaining
counterpart (C and D) were introduced by in situ electroporation into NIH3T3 cells growing on
partly conductive slides (inset, Fig. 1A) and growth arrested in spent medium. Five minutes after pulse application,
cells were stimulated with EGF for 5 rain, fixed, and probed for activated Erk½, and cells from the
same field were photographed under bright-field (A and C) or phase-contrast (B and D) illumination. Magnification:
A and B, 240×, C and D, 40×. Arrow points to the transition line between stripped (b) and electroporated
(a) areas, while arrowhead points to the line between control ITO-coated (c) and stripped (b) areas
(Fig. 1A). Cells growing on the left side (a) are electroporated, whereas cells on the stripped zone (b) or right
side (c) of the slide do not receive any pulse. Note that the Grb2-SH2 blocking peptide dramatically reduced
the EGF signal (A, a), whereas the degree of Erk activation is the same on both sides of the slide (a or c) for
cells electroporated with the control, phenylalanine-containing peptide (C). In A, inhibition of the signal
extends into approximatily three to four rows of adjacent cells in the non-electroporated area (squiggly
bracket in b), probably due to movement of the peptide through gap junctions (Raptis et al., 1994). At the
same time, there is no detectable effect on cell morphology as shown by phase contrast (B and D). From Rapfis et al. (2000), reprinted with permission.
|FIGURE 4 In situ electroporation on a partly conductive slide for the measurement of intercellular,
junctional communication. (A and B) An established, mouse lung epithelial type II line (El0) was plated on
partly conductive slides (inset, Fig. 1B) and at confluence was electroporated in the presence of 5mg/ml
Lucifer yellow. After washing away any unincorporated dye, cells from the same field were photographed
under fluorescence (A) or phase-contrast (B) illumination (Raptis et al., 1994). Note the gradient of fluorescence
indicating dye transfer through gap junctions. To quantitate intercellular communication, the number
of cells into which the dye transferred through gap junctions per electroporated border cell was calculated
by dividing the total number of fluorescing cells on the non-conductive side (white circles) by the number
of cells growing at the border with the conductive coating (black stars). (C and D) A spontaneous transformant
of the El0 line (line E9), was plated on partly conductive slides, electroporated, washed, and photographed
as described earlier. Fluorescence (C) and phase-contrast (D) illumination photograph of the same
field. Note the absence of dye transfer through gap junctions. In all photographs, the left side is conductive.
Arrows on the conductive side point to the interphase between conductive and non-conductive areas. Magnification:
200×. From Vultur et al. (2003), reprinted with permission.
: 5-10mg/ml in calcium-free DMEM.
See Section III,A and Comment
Lucifer yellow solution
: 5 mg/ml in calcium-free DMEM.
See Section III,A
Epidermal growth factor
: 100ng/ml in calcium-free
DMEM. See Section III,A.
- Choice of slides. Use partly conductive slides where
the coating has been removed in a line as shown in
- Plate the cells as described earlier and starve them
- Aspirate the medium and wash the cells once with
- Add the peptide solution as described previously.
- Apply a pulse of the appropriate strength (see Comment 3).
- Add serum-free growth medium and place the cells
in a 37°C incubator for the pores to close (2-5 min).
- Treat the cells with EGF for 5 min as in Section III,A.
- Fix the cells with 4% paraformaldehyde and probe
with the anti-active Erk antibody according to the
As shown in Fig. 3, electroporation of the Grb2-SH2
blocking peptide totally inhibited EGF-induced Erk
activation (panel A, area "a
"), while the control,
phenylalanine-containing peptide had no effect (panel
C, area "a
"). This inhibition was uniform across the cell
layer, in agreement with previous results indicating
that in situ
electroporation can introduce the material
into essentially 100% of the treated cells. It is especially
noteworthy that this inhibition extends into three to
four rows of the adjacent, nonelectroporated cells
growing on the nonconductive part of the slide (panel
A, area "b
", squiggly bracket), probably due to movement
of the 1123Da peptide through gap junctions
(Raptis et al.
, 1994). This finding constitutes compelling
evidence that the observed inhibition must be due to
the peptide rather than an artifact of electroporation. At
the same time, as shown by phase-contrast microscopy
(panel B), there was no alteration in the morphology of
the electroporated cells under these conditions, suggesting
that the observed effect is a result of a specific
inhibition rather than toxic action. EGF stimulation for
up to 30 min after peptide electroporation did not result
in lower levels of Erk signal inhibition, indicating that
the binding of the peptide to Grb2 is stable during this
period of time. As expected, the phenylalanine-containing,
control peptide (panels C and D) had no effect on
Erk activation. In contrast, the Grb2-SH2 binding
peptide had little effect in inhibiting Erk activity triggered by the hepatocyte growth factor (HGF) in
NIH3T3 cells expressing the HGF receptor through
transfection or in human A549 cells that naturally
express this receptor (Raptis et al.
The introduction of peptides to interrupt signaling
pathways using the modification of in situ
described is a powerful approach for the in vivo
assessment of the relevance of in vitro
Results presented in Fig. 2 and 3 clearly demonstrate
that an essentially complete and specific inhibition
of EGF-dependent Erk activation can be achieved
through peptide electroporation. The stepwise dissection
of signaling cascades is essential for the understanding
of normal proliferative pathways, which
could lead to the development of drugs for the rational
treatment of neoplasia.
C. Electroporation on a Partly Conductive
Slide for the Assessment of Gap Junctional,
One of the targets of a variety of signals stemming
from growth factors or oncogenes may be membrane
channels, which serve as conduits for the passage of
small molecules between the interiors of cells. Oncogene
expression and neoplasia invariably result in a
decrease in gap junctional, intercellular communication
(GJIC) (Goodenough et al.
, 1996). The investigation
of junctional permeability is often conducted
through microinjection of a fluorescent dye such as
Lucifer yellow, followed by observation of its migration
into neighboring cells. This is a time-consuming
approach, requiring expensive equipment, while the
mechanical manipulation of the cells may disturb cellto-
cell contact areas, interrupt gap junctions, and cause
artefactual uncoupling. These problems can be overcome
using a setup where cells are grown on a glass
slide, half of which is coated with electrically conductive,
optically transparent, indium-tin oxide. An electric
pulse is applied in the presence of Lucifer yellow,
causing its penetration into cells growing on the
conductive part of the slide, and migration of the dye
to non-electroporated cells growing on the nonconductive
area is observed microscopically under
The technique can be applied to a large variety of
adherent cell types, including primary human lung
carcinoma cells (Tomai et al.
, 1998; Raptis et al.
Brownell et al.
, 1996; Vultur et al.
Lucifer yellow solution
: 5 mg/ml in calcium-free DMEM
or other growth medium. See Section III.A
Calcium-free growth medium with or without 5% dialysed
- Plate the cells on partly conductive slides in 3 cm
petris. Electroporated areas can be 4 × 4mm and
non-electroporated ones 4 × 3 mm (Fig. 1B). Other slide
configurations are also available (Raptis et al., 2000b).
- Aspirate the medium. Wash the cells with
- Add the Lucifer yellow solution.
- Apply a pulse of the appropriate strength so that
cells growing on the conductive coating at the border
with the non-conductive area are electroporated
without being damaged. As described in Comment 3, this area receives slightly larger amounts of current
than the rest of the conductive growth surface.
- Add calcium-free DMEM containing 5% dialysed
serum, remove the electrode, and incubate the cells
for 3-5 min in a 37°C, CO2 incubator. The inclusion of
dialysed serum at this point helps pore closure.
- Wash the unincorporated dye with calcium-free
- Microscopically examine under fluorescence and
phase-contrast illumination (Fig. 4).
- Quantitate intercellular communication. Photograph
the cells with a 20× objective under fluorescence
and phase contrast illumination (Figs. 4A and 4B).
Identify and mark electroporated cells at the border
with the non-conductive area (black stars) and fluorescing cells on the nonconductive side (white circles)
where the dye has transferred through gap junctions.
Divide the total number of fluorescing cells on the
non-conductive area by the number of electroporated
cells along the border with the etched side. The
transfer from at least 200 contiguous electroporated
border cells is calculated for each experiment
(Raptis et al., 1994). A careful kinetic analysis of dye
transfer from 30s to 2h showed that the observed
transfer is essentially complete by 5 min for all lines
tested, while fluorescence is eliminated from the cells
within approximately 60min. After the transfer is
complete, cells can be fixed with formaldehyde,
in which case fluorescence is retained for approximately
The concentration of peptide required varies with
the strength of the signal to be inhibited. For example,
for the inhibition of the HGF-mediated Stat3 activation
in MDCK cells, a concentration of 1 µg/ml of a peptide
blocking the SH2 domain of Stat3 (P
YVNV) is sufficient
(Boccaccio et al.
, 1998), whereas for the inhibition of the
EGF-mediated Erk activation in a variety of fibroblasts
or epithelial cells, a concentration of 5-10mg/ml
(~5-10mM) of the Grb2-SH2 blocking peptide is necessary
(Raptis et al.
, 2000a). The purity of the material
is of utmost importance. Peptides must be HPLCpurified
because impurities can cause cell death or
give unexpected results. The pH of the peptide solution
must be neutral, as indicated by the color of the
DMEM medium where the peptide is dissolved. If it is
too acidic, then it must be carefully neutralised with
NaOH. In this case, the salt concentration of the nopeptide
controls (DMEM without calcium) must be
adjusted to the same level with NaCl because a change
in conductivity may affect the optimal voltage
required (see Comment 3).
Any peptide which is soluble in DMEM or other
aqueous buffer can be very effectively electroporated.
Good solubility is especially important because the
concentration needed for effective signal inhibition can
be as high as 10 mg/ml. It was nevertheless found that,
at least for certain applications, the inclusion of DMSO
in the electroporation solution at a concentration of up
to 5%, which might aid peptide solubility, did not
affect results significantly. However, a number of
peptides, e.g., peptides made as fusions with the
homeobox domain or other membrane translocation sequences, are usually not sufficiently soluble for this
As described in detail in Chapter 43 by Raptis et al.
to obtain a uniform electrical field intensity over the
entire area below the negative electrode, despite the
fact that the conductive coating exhibits a significant
amount of electrical resistance, the bottom surface of
the negative electrode must be inclined relative to the
glass surface in a manner proportional to the resistance
of the coating. For electroporation of peptides, to minimise
the volume of custom-made peptide used, slides
with a conductivity of 2Ω/sq, the most conductive
commercial grade available, are used. The slides and
electrodes come in different sizes, with the biggest cell
growth area in this configuration being 32 × 10mm.
Depending on the experiment, if larger numbers of
cells are required, extracts from two to three slides may
be pooled. In this case, the peptide solution can be
aspirated and used again. Alternatively, an electrode
configuration with two positive contact bars can be
employed, as described in Chapter 43 by Raptis et al.
The slides come with the apparatus, individually
wrapped and sterile. However, they can be reused
many times after washing with Extran-300 detergent
while scrubbing with a toothbrush. In this case they
must be sterilized with 80% ethanol for 20min and
the ethanol rinsed with sterile distilled water prior to
plating the cells. Alternatively, the slides can be gassterilized.
Do not autoclave. The glass can withstand
high temperatures, but autoclaving would damage the
3. Determination of the Optimal Voltage
Electrical field strength has been shown to be a critical
parameter for cell permeation, as well as viability
(Chang et al.
, 1992). It is generally easier to select a discrete
capacitance value for a given electroporated area
and space between the conductive coating and the
negative electrode and then precisely control the
voltage. Both parameters depend upon the size of
the electroporated area; larger conductive growth
areas necessitate higher voltages and/or higher capacitances
for optimal permeation. For the 32 × 10 mm cell
growth area, some damage to the coating may be
noted at the higher voltages necessary if a single pulse
is employed. However, using higher capacitance
values and multiple pulses with lower voltage settings
can yield efficient cell permeation with no damage to the coating, and this treatment is also better tolerated
by the cells. For greater growth areas, the dual positive
contact bar design described in Chapter 43 by Raptis et al.
can be employed.
The optimal pulse strength depends on the strain
and metabolic state of the cells, as well as on the degree
of cell contact with the conductive surface. Densely
growing, transformed cells or cells in a clump require
higher voltages for optimum permeation than sparse,
subconfluent cells, possibly due to the larger amounts
of current passing through an extended cell. Similarly,
cells that have been detached from their growth
surface by vigorous pipetting prior to electroporation
require substantially higher voltages. It is especially
striking that cells in mitosis remain intact under conditions
where most cells in other phases of the cycle
are permeated (Raptis and Firth, 1990). In addition,
cells growing and electroporated on CelTakTM
slides require substantially higher voltages than cells
growing directly on the slide.
The margins of voltage tolerance depend on the size
and electrical charge of the molecules to be introduced
(Fig. 5). For the introduction of small, uncharged
molecules such as Lucifer yellow or peptides, a wider
range of field strengths permits effective permeation with minimal damage to the cells than the introduction
of antibodies or DNA (Raptis and Firth, 1990;
Brownell et al.
, 1997). For a number of experiments
involving cell growth, it may be necessary to electroporate
serum-arrested cells. Voltages required are
lower, and especially the margins of voltage tolerance
were found to be substantially narrower for serumstarved
cells compared to their counterparts growing
in 10% calf serum (Brownell et al.
, 1997). Also, it is
important to keep all solutions at 37°C, which facilitates
pore closure and efficient electroporation. If the
material is applied in a medium with a lower salt concentration
than DMEM, then the voltages required are
lower, presumably due to the hypotonic shock to the
cells and to the longer duration of the pulse because of
the lower conductivity of the medium. Conversely,
electroporation in a hypertonic solution requires
higher voltages for optimum permeation.
|FIGURE 5 Effect of field strength on the introduction of different
molecules. Three pulses of increasing voltage were applied
to confluent rat F111 fibroblasts growing on a conductive surface of
32 × 10mm from a 32µF capacitor in the presence of 5 mg/ml Lucifer
yellow (), 5mg/ml of the Grb2-SH2 blocking peptide (), 5 mg/ml
(), or 100µg/ml pY3 plasmid DNA, coding for resistance
to hygromycin (Raptis and Firth, 1990) (O). Cells were lysed
and Lucifer yellow fluorescence was measured using a Model 204A
fluorescence spectrophotometer (), probed with the anti-active Erk
antibody (), probed for incorporated IgG (), or selected for
hygromycin resistance (O) (Raptis and Firth, 1990). Introduction of
chicken IgG was quantitated from the percentage of cells staining
positive with their respective antibodies. Cell killing () was
assessed by calculating the plating efficiency of the cells after the
pulse. Note that a wider range of voltages (20-50 V) permits efficient
introduction of Lucifer yellow with no detectable loss in cell viability
than the introduction of IgG or DNA. Points represent averages
of at least three separate experiments. L.Y., Lucifer yellow.
Cell damage is manifested microscopically by
the appearance of dark nuclei under phase-contrast
illumination. For most lines this is most pronounced
5-10min after the pulse. Such cells do not retain
Lucifer yellow and fluoresce very weakly, if at all (Fig.
6). Despite the fact that every effort is made to make
the electric field uniform over the whole cell growth
area, the current flow along the border with the etched
side is greater than the rest of the conductive surface.
For this reason, as the voltage is progressively increasing,
damaged cells will appear on this edge first (Fig.
6). Another area receiving a slightly higher current is
corners of the window. This slight irregularity has to
be taken into account when determining the optimal
4. Example of the Determination of Optimal
Voltage for the Introduction of Peptides
|FIGURE 6 Determination of the optimal voltage. Rat F111 fibroblasts growing on partly conductive slides (Fig. 1B, conductive area, 4 × 4
mm) were electroporated in the presence of 5 mg/ml Lucifer yellow using three pulses of increasing voltage delivered from a 0.2µF capacitor.
(A and B) 18 V, (C and D) 26V, (E and F) 28V, (G and H) 32 V, and (I and J) 40V. After washing of the unincorporated dye, cells were photographed
under fluorescence (A, C, E, G, I) or phase-contrast (B, D, F, H and J) illumination. Straight arrow points to the interphase between
conductive (right) and non-conductive (left) areas. Curved arrows in C and D point to a cell which has been killed by the pulse. Note the dark,
pycnotic and prominent nucleus under phase contrast and the flat, nonrefractile appearance. Such cells do not retain any electroporated material
as shown by the absence of fluorescence (C). Note that the number of such cells along the border with the non-conductive area increases
with voltage. Arrowheads in E and F point to a cell that has a prominent nucleus under phase contrast (F) but has retained the dye (E). Such
cells rapidly recover their normal morphology, indistinguishable from their non-electroporated counterparts. White arrowheads in I and J point
to membrane blebs which tend to enclose Lucifer yellow and fluoresce strongly. Such membrane blebbing tends to be more prominent under
higher voltages. Note that if the determination of intercellular communication is desired, then the voltage must be such that cells at the border
with the non-conductive area are electroporated without being damaged (e.g., 18V, A and B), whereas for all other applications, voltages of
approximately 26-32 V would be preferred (C to H). Magnification: A and B, 120X; C-J, 240×.
Prepare a series of slides with cells plated uniformly
in a 4 × 7mm window on a partly conductive slide
(Figs. 1A or 1B). Set the apparatus at 0.5 µF capacitance.
Prepare a solution of 5mg/ml Lucifer yellow and
electroporate at different voltages (0.5 µF, three pulses)
to determine the upper limits where a small fraction of
the cells at the border with the etched side (probably the
more extended ones) are killed by the pulse, as determined
by visual examination under phase-contrast and
fluorescence illumination (Fig. 6). Depending on the
cells, this voltage can vary from 20 to 40 V. Repeat the
electroporation using the peptide solution at different
voltages starting at 10 V below the upper limit and at
2 V increments. The Lucifer yellow dye offers an easy
way to test for cell permeation.
Results of a typical experiment of electroporation of
cells growing on a 32 × 10mm surface are shown in Fig. 5. The Application of three exponentially decaying
pulses of an initial strength of 25V from a 32µF capacitor to rat F111 cells growing on a conductive
growth area of 32 × 10 mm, resulted in essentially 100%
of the cells containing the introduced Lucifer yellow,
whereas introduction of a nine amino acid peptide
required 30 V and the stable expression of DNA 45 V,
respectively, for maximum signal. If a 20µF capacitor
is used, the corresponding voltages are ~80-180. Electroporation
on 7 × 15 mm slides requires a 2µF capacitor
and voltages of 25-50. For etched slides with a conductive area of 4 × 4mm, if a 0.5 µF capacitor and
three pulses are used, the voltages are 20-40. However,
if a 0.1µF capacitor and six pulses are used, the voltages
will be ~30-50.
Under the appropriate conditions, electroporation
was shown not to affect the activity of Erk½ or the
stress-activated kinases JNK/SAPK and p38hog
was shown by probing with antibodies specific for the
activated forms of these kinases (Fig. 7); no activation
of JNK/SAPK or p38hog
was found under conditions
of up to 70V (Figs. 7C and 7E). These kinases were, however, slightly activated at voltages higher than
85 V, when more than 60% of the cells were killed by
the pulse (not shown).
|FIGURE 7 In situ electroporation does not affect ERK activity or the stress pathway. (A, C, and E)
NIH3T3 cells were plated on fully conductive slides (inset, Fig. 1C, conductive growth area 4 × 8mm), growth
arrested in 50% spent medium, and electroporated in the presence of PBS containing 0.025% DMSO (0.2µF,
70 V, four pulses). Ten minutes after the pulse, cells were fixed and stained for activated ERK (A), activated
JNK/SAPK (C), or activated p38hog (E), respectively. Electroporated cells were photographed under brightfield
illumination. (B, D, and F) NIH3T3 cells were plated on conductive slides, treated with UV light for
10rain, fixed, and stained for activated ERK (B), activated JNK/SAPK (D), or activated p38hog (F), respectively.
From Brownell et al. (1998), reprinted with permission. Magnification: 240×.
- Care must be taken so that cells do not dry
during the procedure, especially during wiping of the
frame with a tightly folded Kleenex. It was found that
serum-starved cells were more susceptible than their
counterparts grown in medium containing serum. The
morphology of cells that have been killed by drying is
very similar to cells that have been killed by the pulse
(Fig. 6). Slightly dried cells may incorporate Lucifer
yellow and appear almost normal under phase contrast,
whereas cells that have dried to a great extent
display dark nuclei and may not retain Lucifer yellow.
It was also found that the combination of even slight
drying with electroporation may have undesirable
effects on gene expression (e.g., induction of fos by
serum; unpublished observations). In the case of electroporation
on a partly conductive slide (Figs. 1A or
1B), drying of the cells is immediately suspected if cells
growing on the non-electroporated area exhibit Lucifer
- Accurate determination of the optimal voltage
is very important. The limits of voltage tolerance
are narrower for serum-starved cells (Brownell et al.,
1997) or if the introduction of larger molecules is
- For the determination of GJIC, it is important to
wash the dye using a calcium-free solution (growth
medium or PBS). If calcium-containing growth medium
is used instead, the values obtained may be reduced,
presumably because of the calcium influx, which was
shown to interrupt junctional communication.
The financial assistance of the Canadian Institutes
of Health Research, the Canadian Breast Cancer
Research Initiative, the Natural Sciences and Engineering
Research Council of Canada (NSERC), and the
Cancer Research Society Inc. is gratefully acknowledged.
AV is the recipient of NSERC and Ontario
Graduate studentships, a Queen's University graduate
award, and a Queen's University travel grant. HB was
the recipient of a studentship from the Medical
Research Council of Canada and a Microbix Inc. travel
award. We are grateful to Dr. Erik Schaefer of
Biosource Int. for numerous suggestions and valuable
Bardelli, A., Longati, P., Gramaglia, D., Basilico, C., Tamagnone, L.,
Giordano, S., Ballinari, D., Michieli, P., and Comoglio, P. M.
(1998). Uncoupling signal transducers from oncogenic MET
mutants abrogates cell transformation and inhibits invasive
growth. Proc. Natl. Acad. Sci. USA 95
Boccaccio, C., Ando, M., Tamagnone, L., Bardelli, A., Michielli, P.,
Battistini, C., and Comoglio, P. M. (1998). Induction of epithelial
tubules by growth factor HGF depends on the STAT pathway. Nature 391
Boussiotis, V. A., Freeman, G. J., Berezovskaya, A., Barber, D. L., and
Nadler, U M. (1997). Maintenance of human T cell anergy: Blocking
of IL-2 gene transcription by activated Rap1. Science 278
Brownell, H. L., Firth, K. L., Kawauchi, K., Delovitch, T. L., and
Raptis, L. (1997). A novel technique for the study of Ras activation;
electroporation of [α32
P]GTP. DNA Cell Biol
Brownell, H. L., Lydon, N., Schaefer, E., Roberts, T. M., and Raptis,
L. (1998). Inhibition of epidermal growth factor-mediated
ERK½ activation by in situ
electroporation of nonpermeant
[(alkylamino)methyl]acrylophenone derivatives. DNA Cell Biol
Brownell, H. L., Narsimhan, R., Corbley, M. J., Mann, V. M.,
Whitfield, J. F., and Raptis, L. (1996). Ras is involved in gap
junction closure in mouse fibroblasts or preadipocytes but not in
differentiated adipocytes. DNA Cell Biol
Chang, D. C., Chassy, B. M., Saunders, J. A., and Sowers, A. E. (1992).
"Guide to Electroporation and Electrofusion." Academic Press,
Firth, K. L., Brownell, H. L., and Raptis, L. (1997). Improved procedure
for electroporation of peptides into adherent cells in situ
. Biotechniques 23
Folkman, J., and Moscona, A. (1978). Role of cell shape in growth
control. Nature 273
Gambarotta, G., Boccaccio, C., Giordano, S., Ando, M., Stella, M. C.,
and Comoglio, P. M. (1996). Ets up-regulates met transcription. Oncogene 13
Giorgetti-Peraldi, S., Ottinger, E., Wolf, G., Ye, B., Burke, T. R., and
Shoelson, S. E. (1997). Cellular effects of phosphotyrosinebinding
domain inhibitors on insulin receptor signalling and
trafficking. Mol. Cell. Biol
Goodenough, D. A., Goliger, J. A., and Paul, D. L. (1996). Connexins,
connexons, and intercellular communication. Annu. Rev. Biochem
Kwee, S., Nielsen, H. V., and Celis, J. E. (1990). Electropermeabilization
of human cultured cells grown in monolayers: Incorporation
of monoclonal antibodies. Bioelectrochem. Bioenerg
Marais, R., Spooner, R. A., Stribbling, S. M., Light, Y., Martin, J., and
Springer, C. J. (1997). A cell surface tethered enzyme improves
efficiency in gene-directed enzyme prodrug therapy. Nature
Matsumura, T., Konishi, R., and Nagai, Y. (1982). Culture substrate
dependence of mouse fibroblasts survival at 4°C. In Vitro 18
Nakashima, N., Rose, D., Xiao, S., Egawa, K., Martin, S., Haruta, T.,
Saltiel, A. R., and Olefsky, J. M. (1999). The functional role of crk
II in actin cytoskeleton organization and mitogenesis. J. Biol.
Neumann, E., Kakorin, S., and Toensing, K. (2000). Principles of
membrane electroporation and transport of macromolecules. In "Electrochemotherapy, Electrogenetherapy and Transdermal Drug
(M. J. Jaroszeski, R. Heller, and R. Gilbert, eds.),
pp. 1-35. Humana Press, Clifton, NJ.
Otaka, A., Nomizu, M., Smyth, M. S., Shoelson, S. E., Case, R. D.,
Burke, T. R., and Roller, P. P. (1994). Synthesis and structureactivity
studies of SH2-binding peptides containing hydrolytically
stable analogs of O-phosphotyrosine. In "Peptides; Chemistry,
Structure and Biology"
(R. S. Hodges and J. A. Smith, eds.),
pp. 631-633. Escom, Leiden.
Raptis, L., Brownell, H. L., Firth, K. L., and MacKenzie, L. W. (1994).
A novel technique for the study of intercellular, junctional
communication; electroporation of adherent cells on a partly
conductive slide. DNA Cell Biol
Raptis, L., Brownell, H. L., Vultur, A. M., Ross, G., Tremblay, E., and
Elliott, B. E. (2000a). Specific inhibition of growth factorstimulated
ERK½ activation in intact cells by electroporation of
a Grb2-SH2 binding peptide. Cell Growth Differ
Raptis, L., and Firth, K. L. (1990). Electroporation of adherent cells in situ
. DNA Cell Biol
Raptis, L., Tomai, E., and Firth, K. L. (2000b). Improved procedure
for examination of gap junctional, intercellular communication
by in situ
electroporation on a partly conductive slide. Biotechniques 29
Robinson, M. J., and Cobb, M. H. (1997). Mitogen-activated protein
kinase pathways. Curr. Opin. Cell Biol
Schlessinger, J. (2000). Cell signaling by receptor tyrosine kinases. Cell 103
Tomai, E., Brownell, H. L., Tufescu, T., Reid, K., Raptis, S., Campling,
B. G., and Raptis, L. (1998). A functional assay for intercellular,
junctional communication in cultured human lung carcinoma
cells. Lab. Invest
Vultur, A., Tomai, E., Peebles, K., Malkinson, A. M., Grammatikakis,
N., Forkert, P. G., and Raptis, L. (2003). Gap junctional, intercellular
communication in cells from urethane-induced tumors in
A/J mice. DNA Cell Biol
Williams, E. J., Dunican, D. J., Green, P. J., Howell, F. V., Derossi, D.,
Walsh, F. S., and Doherty, P. (1997). Selective inhibition of growth
factor-stimulated mitogenesis by a cell-permeable Grb2-binding
peptide. J. Biol. Chem
Yang, T. A., Heiser, W. C., and Sedivy, J. M. (1995). Efficient in situ
electroporation of mammalian cells grown on microporous membranes. Nucleic Acids. Res