Cytotechnology (2014) 66:543–553

DOI 10.1007/s10616-013-9608-9

TECHNICAL NOTE

An electroporation protocol for efficient DNA transfection

in PC12 cells
Giuseppina Covello • Kavitha Siva •
Valentina Adami • Michela A. Denti

Received: 6 April 2013 / Accepted: 14 June 2013 / Published online: 12 July 2013
Ó The Author(s) 2013. This article is published with open access at Springerlink.com

Abstract A wide variety of mammalian cell types is parameters (voltage, pulse width and number of
used in gene transfection studies. Establishing trans- pulses) we reached high efficiency of transfection
fection methods that enable highly efficient DNA (90 %) and viability (99 %). We also demonstrated
uptake has become increasingly important. PC12 is an that, upon electroporation, cells are not altered by the
established rat pheochromocytoma cell line, which transfection and maintain their ability to differentiate.
responds to exposure to NGF with cessation of growth,
expression of cytoplasmic processes, and differentia- Keywords PC12 cells
Cell culture
DNA
tion into cells resembling sympathetic neurons. transfection
DNA electroporation
NGF
Neural
Although PC12 cells represent an important model differentiation
system to study a variety of neuronal functions, they
proved relatively difficult to transfect. We have
compared the efficiency of three different chemical Introduction
transfection reagents (Lipofectamine 2000, Lipofect-
amine LTX and TransIT-LT1) and of two electropor- PC12 cells are a cell line originating from pheochro-
ation systems (Neon and Gene Pulser Xcell) in mocytoma in the rat adrenal medulla (Schaefer et al.
transiently transfecting undifferentiated PC12 cells. 1987) and grow in culture as undifferentiated neuro-
By comparing efficiencies from replicate experiments blasts. Since its characterization in 1976 (Greene and
we proved electroporation (in particular Neon) to be Tischler 1976) PC12 cells have become a commonly
the method of choice. By optimizing different employed model system for studies of neuronal
development and function (Grau and Greene 2012).
One of the important features of PC12 cells is that they
G. Covello
K. Siva
M. A. Denti (&)
Laboratory of RNA Biology and Biotechnology, Centre are small cells with a limited amount of cytoplasm and
for Integrative Biology (CIBIO), University of Trento, long doubling time. They possess remarkable ability to
via delle Regole 101, 38123 Trento, Italy respond to nerve growth factor (NGF), a neurotrophic
e-mail: denti@science.unitn.it
polypeptide, inducing morphological and biochemical
V. Adami changes resulting in differentiation of PC12 cells into
High-Throughput Screening Facility, Centre for a sympathetic neuron-like phenotype (Greene and
Integrative Biology (CIBIO), University of Trento, Tischler 1976; Grau and Greene 2012; Nagase et al.
via delle Regole 101, 38123 Trento, Italy
2005; Dhar et al. 2007; Park et al. 2007). For this
M. A. Denti reason PC12 cells have been regarded as a research
CNR Institute of Neuroscience, Padua, Italy model to study neuronal development, sympathetic

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neurotransmission and neurodegenerative diseases (Invitrogen) the transfection efficiency was about
(Wang et al. 2002; Seth et al. 2002). In general, when 14 % and was similar to the efficiency obtained with
challenged with physiological levels of NGF, these polyethyleneimine (PEI) (15 %) (Lee et al. 2008).
cells cease division, become electrically excitable, A higher efficiency (30 %) was reported with Meta-
extend long branching neurites, and gradually acquire fectene Pro (Biontex) (Cogli et al. 2010). An increase
many characteristics of mature sympathetic neurons. in the transfection efficiency (40–50 %) was observed
Under serum-free conditions, NGF promotes not only by simultaneous treatment with Lipofectamine and
neuronal differentiation of PC12 cells, but also their 0.1 lM GALA, a pH-sensitive fusogenic peptide
survival (Greene 1978; Fujita et al. 1989; Rukenstein which accelerates the endosomal escape of the plas-
et al. 1991). Several of their attributes have led to their mid/liposome complexes to the cytosol (Futaki et al.
widespread popularity in neurobiological research. 2005). However, this method has not encountered
These include their relatively high degree of differen- wide popularity thereafter.
tiation before and after NGF treatment, homogeneous Physical methods, including electroporation, bio-
response to stimuli, availability in large numbers for listics and injection, are used with varying success and
biochemical studies, and suitability for genetic manip- are cell cycle-independent but may be more toxic for
ulations. However, finding transfection techniques some cell types and usually require cell suspensions
that enable efficient DNA uptake into PC12 cells is in vitro and specialized equipment (Villemejane and
relatively difficult. Moreover, it is important to have a Mir 2009). However, the electroporation methods
good method to transfect these cells with high enable efficient transfer of exogenous DNA to a large
efficiency devoid of cellular alteration, and maintain- number of cells and serve ideal in terms of material
ing their ability to differentiate. An important limita- and time consumption. As with chemical methods of
tion of working with PC12 cells is that they tend to be transfection, high-efficiency electroporation protocols
very sensitive to physical stress, alterations in tem- for PC12 cells are not available. The literature reports
perature, pH shifts, or changes in osmolarity. There- efficiencies between 10 and 20 % (Darchen et al.
fore, handling and manipulation during transfection is 1995; Akamatsu et al. 1999) and 35 % (Lombardi
a crucial step. et al. 2001), which increases to 50 % when parameters
Various transfection methods have been attempted are carefully optimized (Espinet et al. 2000).
to transfect this cell line. In general, cells can be gene- The aim of this work was to find a protocol ensuring
modified in vitro and in vivo using chemical or physical high transfection efficiency in PC12 cells, while
methods (Azzam and Domb 2004; Marples and Dachs retaining viability and ability to differentiate. We
2002). Chemical methods of transfection are widely compared two electroporation systems (Neon trans-
used, as they are relatively simple, cheap, and safe fection and Gene Pulser Xcell) and three chemical
(Douglas 2008). They include calcium phosphate, transfection methods (Lipofectamine 2000, Lipofect-
liposomes, cationic lipids (e.g., dioleoyl trimethylam- amine LTX, TransIT-LT1).
monium propane (DOTAP)), cationic polymers (e.g.,
polyethylenimine (PEI), dendrimers) and cationic
polysaccharides (Eliyahu et al. 2005; Godbey et al. Materials and methods
1999a, b). In general, these reagents act via packaging
mechanism to condense and deliver DNA to the Plasmid
cytoplasm of cells, usually by endocytosis (Vijayana-
than et al. 2002). These reagents are used rapidly in DNA plasmid used for transfection or electroporation
high-throughput assays and can transfer DNA of was pEGFP-C1 (BD Biosciences Clontech, Palo Alto,
various sizes (Ewert et al. 2008). However, they can CA, USA) driving the expression of an enhanced green
be susceptible to nuclease degradation, are potentially fluorescent protein (EGFP) under the control of the
harmful (Colombo et al. 2001) and are usually, but not CMV promoter. Plasmid was amplified in Escherichia
always, cell cycle-dependent (Brunner et al. 2002). coli DH5a; it was isolated and purified using Endo-
In the particular case of PC12 cells, cationic lipids Maxi Free Kit from QIAGEN. DNA purity and
formulations have been employed to increase integrity were determined spectroscopically (OD260nm/
transfection efficiency. Using Lipofectamine 2000 OD280nm = 1.90–2.00), (OD260nm/OD230nm [ 2.00).

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Culture of PC12 cells For the electroporation with the Neon System, PC12
cells were grown to 70 % confluence in a poly-D-
Rat PC12 cells (ATCC entry CRL-1721) were grown lysine-coated T-25 flask and washed twice with
at 37 °C (5 % CO2) in supplemented DMEM: Dul- 10 ml Phosphate-Buffered Saline without Ca2? and
becco’s modified Eagle’s medium with 4.5 % glucose Mg2?(PBS) (Lonza). This was followed by addition of
(Lonza, Visp, Switzerland) supplemented with 10 % 1 ml 19 trypsin (Lonza) and incubation for 2 min at
fetal bovine serum (Gibco, Grand Island, NY, USA), 37 °C. After adding 9 ml of supplemented DMEM, the
5 % horse serum (Gibco), 1 mM glutamine (Gibco), cells were resuspended, transferred in a 15 ml polypro-
1 mM Penicillin/Streptomycin (Gibco). Cells were pylene tube (Sarstedt, Verona, Italy) and centrifuged at
seeded onto T-75 cm2 flasks (Corning, NY, USA) 2.2009g for 10 min. The pellet was resuspended in
coated with 50 ng/ml poly-D-lysine hydrobromide 10 ml of PBS and cells were counted in a Bürker
(Sigma, St. Louis, MO, USA), to achieve 70 % chamber (PAUL MARIENFELD GmbH). Cells were
confluence. Cells from passages 8–10 were used. pelleted again and re-suspended in Resuspension Buffer
Cells were split every other day at a ratio of about 2:3. R (Neon 10 ll kit Invitrogen) to a final concentration
A Pasteur pipette was used to de-aggregate cell of 1 9 107 cells ml-1. 0.6 9 105 and 1 9 105 cells
clusters. were transferred to a sterile 1.5 ml microcentrifuge tube
(Sarstedt), brought to 10 ll final volume of cell
Viable cells counts suspension with Buffer R, and mixed with 500 ng of
pEGFP-C1 vector. To optimize the best condition of
A Trypan Blue Stain exclusion test (Invitrogen, transfection, electroporation was then carried out with
Carlsbad, CA, USA) was used to distinguish viable different voltage, pulse and time parameters, according
from nonviable cells. The suspended cells (9 ll) were to manufacturer’s instructions, as reported in Results
mixed with 1 ll of 0.4 % Trypan Blue Stain and and Discussion. Cells were seeded in a 24- well poly-D-
analyzed in the CountessTM Automated Cell Counter lysine-coated cell plate with 0.5 ml of pre-warmed
(Invitrogen) chamber slide. The percentage of viable supplemented DMEM without antibiotics.
cells was calculated as follows: (number of viable For the electroporation with the Gene Pulser Xcell
cells/total number of cells) 9 100. System, PC12 cells were grown to 70 % confluence in a
poly-D-lysine-coated T-75 flask and washed and tryp-
Lipid-mediated DNA transfection sinized as described above. After adding supplemented
DMEM, the cells were resuspended, transferred in a
The transfection reagents used in this study were: polypropylene tube and centrifuged at 4009g for
TransIT-LT1 Transfection Reagent (Mirus, Madison, 5 min. The pellet was resuspended and counted as
WI, USA), Lipofectamine 2000 and Lipofectamine above, and re-suspended in an appropriate amount of
LTX (Invitrogen). PBS. 0.4 ml of cell suspension containing 6 9 105 or
4 9 104 cells/well were seeded on poly-D-lysine- 1 9 106 cells and 8 lg of DNA (pEGFP-C1 vector),
coated 24-well plates (Corning) one day before trans- were incubated on ice for 10 min, and transferred into a
fection and grown in supplemented DMEM at 37 °C 4-mm electroporation cuvette (BTX). The Gene Pulser
and 5 % CO2. PC12 cells grown to 70 % confluence Xcell System (BIORAD) was used for single-cuvette
were transfected with the mammalian expression electroporation. Electroporations were carried out with
vector pEGFP-C1. Transfection procedures were different voltage and capacitance parameters, accord-
performed as indicated by manufacturer. The ratio ing to manufacturer’s instructions, as reported in
DNA (lg) : transfection reagent (ll) was always 1:3. Results and Discussion. Cells were seeded in a 12-
well poly-D-lysine-coated cell plate with 1 ml of pre-
Transfection by electroporation warmed supplemented DMEM without antibiotics.

DNA electroporation was performed with the NeonÒ High-content image acquisition and analysis
Transfection System MPK5000 (Invitrogen) or the
Gene Pulser Xcell System (BIORAD, Hercules, CA, Upon chemical transfection or electroporation of pEG-
USA). FP-C1 vector, EGFP expression and cell nuclei were

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visualized using Operetta High Content Imaging Sys- Italy) and a 20X objective (magnification). Filter A
tem (PERKIN ELMER, Monza, Italy). To count total (Exciter BP340–380 nm; Dichromatic Mirror 400 nm)
cell numbers, nuclei were counterstained with Hoechst and I3 (Exciter BP450–490 nm; Dichromatic Mirror
33342 (Invitrogen). Forty-eight hours after transfection, 510 nm) were used and the images were analysed with
cells were washed once with PBS and incubated in ImageJ software (http://imagej.nih.gov/ij/) to determine
0.5 ml of supplemented DMEM containing 1 mg/ml of the neurite length (ImageJ plugin NeuronJ) and to
Hoechst 33342, for 20 min at 37 °C and 5 % CO2. Cells quantify the percentage of differentiated cells. A neurite
were washed once with PBS and replaced with supple- was defined as a process of length equal to or greater
mented DMEM without phenol red (Gibco) for imaging. than one time the diameter of the cell body. Triplicate
Images were acquired on an Operetta System using a wells were used routinely for each experimental con-
209 LWD objective in wide-field mode in combination dition. Images of three different fields were taken per
with filters for Hoechst 33342 (excitation filter: well. The experiment was repeated two times.
360–400 nm; emission filter: 410–480 nm) and Alexa
Fluor 488 (excitation filter: 460–490 nm; emission Statistical analysis
filter: 500–550 nm). The laser autofocus was applied
and 10 image fields were acquired per well. For All statistical analyses were performed using Graph-
quantitative analyses, individual cells were segmented pad Prism software package (GraphPad Software, San
based on the Hoechst 33342 nuclear stain using the Find Diego, CA, USA). Student’s t test and One-way
Nuclei building block in the HarmonyÒ High Content analysis of variance (ANOVA) with Bonferroni’s
Imaging and Analysis Software (PERKIN ELMER) and multiple comparison were adopted. A single level of
GFP intensity was quantified within the Hoechst- 0.05 (*p \ 0.05) was used, unless otherwise stated.
defined boundaries for each cell. The Select Population Data are shown with the standard error of the mean
module of Harmony allowed to set a fluorescence (mean ± SEM, n = 3).
intensity threshold in order to identify the sub-popula-
tion of transfected cells and to determine the transfection
efficiency. The average and standard error mean (SEM) Results and discussion
were calculated from biological experimental triplicates
and technical duplicates. Comparison of transfection chemical methods
in PC12 cells
Cell differentiation
We compared transfection efficiencies obtained in
Electroporated PC12 cells were plated at a density of PC12 cells with the lipopolyplex transfection reagent
1 9 105 cells/well on poly-D-Lysine-coated 12-well TransIT-LT1 (Mirus) and the cationic lipids Lipofect-
plates and grown in supplemented DMEM without amine 2000 and LTX (Invitrogen). Cells were grown
antibiotics at 37 °C in 5 % CO2. After 24 h the medium in conditions promoting proliferation including use of
was replaced with differentiation medium (Dulbecco’s an enriched medium, but the transfections were carried
modified Eagle’s medium with 4.5 % glucose supple- out in a serum- and antibiotic- free environment. We
mented with 0.3 % fetal bovine serum, 0.7 % horse performed these experiments with different amounts
serum, 1 mM glutamine) containing 75 ng/ml of NGF of pEGFP-C1 DNA (Clontech), a plasmid driving the
2.5S (Invitrogen). The cells were maintained at 37 °C expression of an enhanced green fluorescent protein
and 5 % CO2 and the differentiation medium was (EGFP) under the control of the CMV promoter. The
replenished every 2nd day for 7 days. ratio DNA (lg): transfection reagent (ll) was 1:3 and
we transfected increasing amounts of plasmid (0.25,
Neurite analysis 0.5, 0.75 and 1 lg).
Forty-eight hours after transfection, nuclei were
Neuronal differentiation was estimated every day, for stained with the viable Hoechst 33342 fluorescent dye
7 days, after exposure to NGF 2.5S, by measurement of and images were acquired on an Operetta System,
morphological parameters. Images were acquired using which combines fluorescence microscopy in a multi-
a fluorescence microscope (DFC420C, Leica, Milan, well format with automated image acquisition and

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quantitative analysis (Fig. 1). Data analysis for trans- In particular, comparing our results with those by
fection efficiency was performed by using the Har- Lee and colleagues (2008), with Lipofectamine 2000
monyÒ High-Content Imaging Software (Perkin Elmer), we reached 21 % transfection efficiency (with 0.5 lg
comparing the number of EGFP-positive cells (detected of DNA in a 24-well plate) while Lee and collabora-
with an Alexa Fluor 488 filter) to the total number of cells tors reported 14 % efficiency in similar conditions
(detected with an Hoechst 33342 filter). Mock transfec- (1 lg of DNA in a 12-well plate).
tion controls using the transfection reagent without DNA Cell viability was measured by Trypan Blue
had an auto-fluorescence background in both the Hoe- Staining 48 h after transfection (Fig. 2b). Mock-
chst 33342 or Alexa Fluor 488 channels comparable to transfected cells had a viability comparable to the
un-transfected PC12 cells (data not shown). non-transfected cells with either of the three transfec-
As shown in Fig. 2a, the TransIT-LT1 Transfection tion reagents (approx. 95 %). For all methods, viabil-
Reagent (Mirus) was not effective in facilitating ity decreased as the DNA amounts increased.
transfection of DNA in PC12 cells, even when a higher However, as clearly indicated in Fig. 2b, there is a
concentration of DNA was used. However, in parallel variation of trend for different reagents. TransIT-LT1
experiments performed on the HEY4 ovarian cancer had a milder impact on cell viability, reaching 92 %
cell line, TransIT-LT1 had a transfection efficiency of when 1 lg of DNA was used. On the contrary,
approximately 35 %, thereby indicating that efficiency Lipofectamine 2000 and Lipofectamine LTX reached
depends on the cell type (data not shown). 87–88 % viability in those conditions.
On the contrary, when we performed the transfec-
tion experiments by using Lipofectamine LTX and DNA electroporation in PC12 cells
Lipofectamine 2000 we managed to transfect DNA
into PC12 cells, with efficiencies that ranged from 7 As the transfection efficiency obtained with Lipofect-
and 15 % respectively, with 0.25 lg DNA, to 35 and amine 2000 was not sufficient for our purposes, we
46 % respectively, when 1 lg of DNA was used. investigated if we could obtain a higher percentage of
As expected, transfection efficiency for both transfected PC12 cells with an electroporation method
reagents correlates with the amounts of DNA used. (Neon Transfection System, Invitrogen).
However, in comparing the transfection efficiency of To optimize conditions, 0.5 lg of plasmid DNA
the two cationic lipids, Lipofectamine LTX seems to and two different cell densities (6 9 104 or 1 9 105
perform better than Lipofectamine 2000 at low DNA cells/well) were used. Moreover, a range of voltage,
amounts, while Lipofectamine 2000 outperforms pulse width and pulse number combinations were
Lipofectamine LTX at higher DNA amounts. tested (Table 1).

Fig. 1 Transfection of rat PC12 cells. PC12 cells were were analyzed by High-Content screening system (Operetta) 48
transfected by liposoluble agents (TransIT-LT1 Transfection h after transfection. The panel shows photographs taken with a
Reagent [Mirus], Lipofectamine LTX [L_LTX] and Lipofect- green filter (left) and a blue filter (center), and the merged
amine 2000 [L_2000]) and different concentrations of a plasmid images (right). Original magnification 920 (Scale
encoding EGFP. Fluorescence images of transfected PC12 cells bars = 200 lm). (Color figure online)

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Fig. 2 Transfection efficiency and viability of rat PC12 cells. viable cells after transfection was measured by a Trypan Blue
a The percent of transfected cells was calculated by dividing the assay. The best cell viability (93.6 %) was obtained with 0.5 lg
number of EGFP positive cells by the total population. The best of plasmid and Lipofectamine 2000. Data represent mean ±
result was obtained by using 1 lg of plasmid and Lipofectamine SEM obtained from triplicates
2000 (efficiency of transfection 45.9 %). b The percentage of

Forty-eight hours after transfection, nuclei were that one obtained by the use of Lipofectamine 2000
stained with Hoechst 33342 and images were acquired (dashed line in Fig. 4a).
on an Operetta System (Fig. 3). Data analysis of The method shows high reproducibility, as deter-
transfection efficiency was carried out as detailed mined by comparing efficiencies from several repli-
above and revealed that the density of the cells in the cate experiments.
suspension is one of the most important variables Cell viability was measured by Trypan Blue Stain-
affecting transfection efficiency in our electroporation ing 48 h after transfection (Table 1). Mock-electropo-
protocols (Fig. 4a). In fact, with higher cell density, rated cells were manipulated as the electroporated cells
any electroporation condition tested yields high but did not receive DNA nor an electrical pulse, and
transfection efficiency, ranging between 80 and had a viability comparable to the non-electroporated
98 %. On the contrary, when a lower cell density cells (approx. 90 %). In the case of cell viability, there
was used, conditions can be separated into two classes, was no clear correlation with cell density, although in
based on the effect they have on transfection effi- general cells at higher density performed better
ciency: a high-efficiency class (84–91 %) and a low- (Fig. 4b). Furthermore, different electroporation con-
efficiency class (20–66 %). ditions worked better for the different cell densities,
It has to be pointed out, however, that all condi- and a general trend could not be inferred from the graph
tions, except one (6 9 104 cells, 1,500 V, 20 ms, 1 in Fig. 4b. In general, the electroporation protocol
pulse), yielded a transfection efficiency higher than appeared to be more pernicious to PC12 cells than the

Table 1 Parameters for DNA electroporation by Neon transfection System, transfection efficiency and cell viability
Pulse voltage (V) Pulse width (ms) Pulse no. Transfection Efficiency (%) Cell viability (%)
0.6 9 105 1 9 105 0.6 9 105 1 9 105
cells/well cells/well cells/well cells/well

Control without electroporation – – 90.00 90.00

1,500 20 1 20.33 93.94 63.30 86.70
1,400 30 1 64.37 80.97 38.40 67.70
1,500 10 3 66.20 90.37 54.70 99.00
1,400 10 3 84.63 87.07 64.20 79.70
1,300 30 1 84.37 97.83 86.70 62.87
1,000 40 1 91.32 94.03 24.90 51.70

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Fig. 3 Representative
images of PC12 cells 48 h
after electroporation.
Electroporation was carried
out using different
conditions of voltage (1000,
1300, 1400 or 1500 Volts),
pulse width (10, 20, 30 or 40
ms) and pulse number (1 or 3
pulses), as indicated on the
left side of the panels.
Pictures were taken with the
high-content operetta
system with a blue filter
(Hoechst) and a green filter
(EGFP), and merged
(MERGE). Original
magnification: 920 (Scale
bars = 50 lm). (Color
figure online)

lipofection protocols, as only few conditions provided viability is concerned, though, Gene Pulser Xcell
a cell viability comparable or higher than that one compared to lipofection (Fig. 4d) and was better than
obtained by Lipofectamine LTX and 1 lg of DNA Neon System in the majority of conditions.
(dashed line in Fig. 4b). For our purposes, the best electroporation condi-
In order to establish if electroporation in general is a tions were obtained with Neon transfection System,
better method than lipofection, or if the Neon System in 1 9 105 cells/well and 3 pulses of 1,500 V and 10 ms
particular has a high performance in transfecting PC12 each. These conditions, in fact, yielded 90 % trans-
cells, we performed electroporation with a different fection efficiency and 99 % viability. We have chosen
system, namely Gene Pulser Xcell (Bio-Rad). 8 lg of these conditions for the subsequent experiments.
plasmid DNA and two different cell densities (6 9 105
or 1 9 106 cells/well) were used. The increase in DNA Cell differentiation and neurite analysis
and cell amounts, compared to Neon System, is due to
the fact that, while the Neon system is miniaturized to With the aim of investigating whether the electropo-
use electroporation tips and 10 ll transfection volumes, rated PC12 cells retain the ability to differentiate into
the Gene Pulser XCell requires specific 400 ll cuvettes. neuron-like cells, we transfected the cells using the
For each cell density we tested three different voltage conditions reported above, and treated them with NGF
and capacitance combinations (Table 2), in triplicate 2.5S (75 ng/ml) starting 24 h after transfection (day 0)
experiments. Namely, we tried 240 V/1,000 lF (Mur- and for the subsequent 7 days (Fig. 5a).
phy et al. 2008), 250 V/960 lF (Yaron et al. 2001) and Images were taken at 8 h and every 24 h after
300 V/500 lF (Lombardi et al. 2001). Analysis of the transfection, in bright field and with a green fluores-
transfection efficiency and cell viability was carried out cence filter (Fig. 5b). NGF-treated PC12 cells showed
as described above. As visible in Fig. 4c and in Table 2, increase in neurite length and generation during time
in our hands the Gene Pulser Xcell system outper- (Fig. 5b). The total number of cells and the number of
formed lipofection methods, reaching efficiencies cells presenting neurites was counted in the bright-
higher than the ones reported in the literature. However, field images. Differentiated PC12 cells progressively
when comparing Gene Pulser Xcell with Neon System, increased reaching 23 % of total cells 4 days after
the latter remained the method of choice for the induction. At later times the percentage of differen-
transfection of PC12 cells (Fig. 4a, c). As far as tiated cells remained stable (Fig. 5c).

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Fig. 4 Optimization of PC12 cells transfection by electropor- electroporation was measured by Trypan Blue Staining. The
ation. Electroporation was carried out in different conditions best cell viability (97.8 %) was obtained with 0.5 lg of plasmid,
which are indicated on the panels’ x-axes in the format ‘‘voltage 1 9 105 cells/well, and 3 pulses of 1,500 Volt and 10 ms each.
(V)/pulse width (ms)/pulse number’’ for panels A and B, and in Dashed line represents worst viability percentage obtained in
the format ‘‘pulse voltage (V)/capacitance (lF)’’ for panels C lipofection experiments as shown in Fig. 2. c Transfection
and D. a Histograms of percentages of transfected PC12 cells efficiency of electroporation experiments performed using Gene
show an increase of efficiencies comparing Neon electropora- Pulser Xcell. Dashed line represents the best transfection
tion with lipofecting agents. Dashed line represents the best efficiency obtained in lipofection experiments as shown in
transfection efficiency obtained in lipofection experiments as Fig. 2. d Viability of cells electroporated with Gene Pulser
shown in Fig. 2. The best condition (99 %) was obtained by Xcell. Dashed line represents worst viability percentage
0.5 lg of plasmid, 1 9 105 cells/well, and 1 pulse of 1,300 Volt obtained in lipofection experiments as shown in Fig. 2. Data
and 30 ms. b The percentage of viable cells after Neon represent mean ± SEM obtained from triplicates

Table 2 Electroporation
Pulse voltage (V) Capacitance (lF) Transfection efficiency (%) Cell viability (%)
parameters applied for
6 6
DNA transfection by Gene 0.6 9 10 1 9 10 0.6 9 106 1 9 106
Pulser Xcell, transfection cells/well cells/well cells/well cells/well
efficiency and cell viability
Control without electroporation – – 98.22 97.94
240 1,000 46.54 54.01 97.08 95.98
250 960 49.56 73.58 94.88 96.56
300 500 63.44 62.98 90.42 91.91

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Fig. 5 Differentiation of electroporated PC12 cells. a Time transfection and differentiation. Cells were grouped in four
course of PC12 differentiation. At each indicated time point, categories: green, differentiated cells; green, non-differentiated
pictures were taken in bright field and in green fluorescence, and cells; non-green, differentiated cells; non-green, non-differen-
cells were counted. b Representative images of PC12 cells tiated cells. Data represent images of three different fields taken
treated with NGF for the indicated time. Original magnification: per each well. The experiment was repeated two times. (Color
920 (Scale bars = 50 lm). c Histogram of cell numbers after figure online)

EGFP expression was observed both in differenti- point, the intracellular expression of EGFP in NGF-
ated and non-differentiated cells. The intensity of differentiated PC12 cells was located in the nucleus.
EGFP signal increased over time. At the initial time At later time points, it was around cytoplasm and

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inside the nucleus. On the 7th day post-transfection, References

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Acknowledgments This work was supported by ‘‘Fondazione Health Perspect 80:127–142
Telethon’’ (Grant GGP08244), by Italian Ministry of Health, Futaki S, Masui Y, Nakase I, Sugiura Y, Nakamura T, Kogure
Young Investigators 2008 (Grant GR-2008-1136933) and by K, Harashima H (2005) Unique features of a pH-sensitive
Italian Ministry of Education, University and Research, ‘‘Futuro fusogenic peptide that improves the transfection efficiency
in Ricerca’’ (Grant RBFR-0895DCB). We thank Dr. Margherita of cationic liposomes. J Gene Med 7(11):1450–1458
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Conflict of interests The authors declare no competing cellular path of poly (ethylenimine)/DNA complexes for
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Grau CM, Greene LA (2012) Use of PC12 cells and rat superior
Open Access This article is distributed under the terms of the cervical ganglion sympathetic neurons as models for neu-
Creative Commons Attribution License which permits any use, roprotective assays relevant to Parkinson’s disease. Meth-
distribution, and reproduction in any medium, provided the ods Mol Biol 846:201–211
original author(s) and the source are credited.

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