Methods 33 (2004) 95103

www.elsevier.com/locate/ymeth

Advanced transfection with Lipofectamine 2000 reagent:

primary neurons, siRNA, and high-throughput applications
Brian Dalby,a,* Sharon Cates,a Adam Harris,a Elise C. Ohki,a Mary L. Tilkins,b
Paul J. Price,b and Valentina C. Ciccaronec
b

a
Invitrogen Corporation Research and Development, 1610 Faraday Avenue, Carlsbad, CA 92008, USA
Invitrogen Corporation, GIBCO Cell Culture Division, 3175 Staley Road, Grand Island, NY 14072, USA
c
MacroGenics, Inc., 1500 East Gude Drive, Rockville, MD 20850, USA

Abstract
Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection eciency and high levels of transgene
expression in a range of mammalian cell types in vitro using a simple protocol. Optimum transfection eciency and subsequent cell
viability depend on a number of experimental variables such as cell density, liposome and DNA concentrations, liposomeDNA
complexing time, and the presence or absence of media components such as antibiotics and serum. The importance of these factors
in Lipofectamine 2000 mediated transfection will be discussed together with some specic applications: transfection of primary
neurons, high throughput transfection, and delivery of small interfering RNAs.
2003 Elsevier Inc. All rights reserved.

1. Introduction
A very large number of techniques for the transfection of mammalian tissue culture cells have been
described. The general requirements for a useful transfection reagent are that it is applied using simple, robust
protocols to eect ecient transfection in a wide range
of cell types and transfection formats, without excessive
cytotoxicity. In this review, we discuss the requirements
for successful transfection and the potential for optimization of transfection eciency using Lipofectamine
2000.
For successful transfection a nucleic acid, which
carries a net negative charge under normal physiological
conditions, must come into contact with a cell membrane that also carries a net negative charge. Lipofectamine 2000 is a cationic liposome formulation that
functions by complexing with nucleic acid molecules,
allowing them to overcome the electrostatic repulsion of
the cell membrane and to be taken up by the cell.
While there is a great deal of structural variation in
lipid molecules that are able to mediate transfection, all
*
Corresponding author. Fax: 1-760-476-6846.
E-mail address: brian.dalby@invitrogen.com (B. Dalby).

1046-2023/$ - see front matter 2003 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymeth.2003.11.023

have a number of common features [1]. First, there is a

positively charged head group that usually contains one
or more positively charged nitrogen atoms to allow interaction between the transfection reagent and the negatively charged sugarphosphate backbone of a nucleic
acid molecule. A spacer usually links the charged head
group to one, two or three hydrocarbon chains. In some
instances, this spacer may play a role in promoting
contact between the cationic lipid and the nucleic acid.
The hydrocarbon chains are often 14 or more carbon
atoms in length. The degree of saturation and the
presence of cis- or trans-forms introduce more potential
for structural variations in these molecules.
The cationic lipid molecule is often formulated with a
neutral co-lipid (helper lipid). Ideally a mixture of the
two lipids is treated by a physical cavitation method
such as sonication or microuidization resulting in the
formation of unilamellar liposomes of relatively uniform
size (typically 100 nm in diameter). The positive charge
on the surface of the liposome generates an electrostatic
interaction with nucleic acids and facilitates contact
with the negatively charged cell membrane. The neutral
co-lipid mediates fusion of the liposome with the cell
membrane eecting entry of the nucleic acid. To achieve
expression of the transgene, DNA must reach the

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B. Dalby et al. / Methods 33 (2004) 95103

nucleus of the cell and become accessible to the transcriptional machinery. In actively dividing cells, transfected DNA may simply become trapped in the nucleus
following the reassembly of the nuclear envelope at the
end of mitosis [24]. However, Lipofectamine 2000 efciently transfects post-mitotic neurons [5] and rat primary hepatocytes as well (S. Cates and B. Dalby,
unpublished observations), suggesting that Lipofectamine 2000 may promote penetration of DNA through
intact nuclear envelopes in these cell types, further
demonstrating its utility.

2. Considerations for optimal transfection using Lipofectamine 2000

2.1. Protocol 1: transfection procedure for 24-well plate
format

for transgene expression or selection for stable transfection. It is not necessary to remove the DNAlipid
complexes nor to change the medium; however, medium may be replaced after 46 h without loss of
transfection activity.
7. Assay for b-galactosidase activity by ortho-nitrophenyl-D -galactopyranoside (ONPG) hydrolysis
using the Invitrogen b-Gal Assay kit (Catalog No.
K1455-01) or similar. Activity can be quantitated
by absorbance measurement at 405 nm using a Molecular Devices SpectraMax 250 microplate reader
or similar. Alternatively b-galactosidase activity can
be measured by using a chemiluminscence assay, such
as the Tropix Galacto-Star System (Applied Biosystems, Catalog No. BM100S). Luminescence can be
measured using a 96-well luminometer (EG&G Berthold Microplate luminometer LB96V or similar).
2.2. Cell density

1. For adherent cells: one day before transfection, seed

0.52
105 cells per well in 500 ll of growth medium
without antibiotics to attain 9095% conuence at
the time of transfection. For suspension cells: on
the day of transfection, just prior to preparing complexes, seed 48
105 cells per well in 500 ll of
growth medium without antibiotics.
2. For each transfection sample, prepare DNALipofectamine 2000 complexes as follows: dilute 0.8 lg
DNA in 50 ll Opti-MEM I (Invitrogen Catalog No.
31985) without serum (or other medium without serum). Mix gently. In the experiments described below, a plasmid containing a b-galactosidase reporter
gene, pCMV.SPORT, is used but transfection parameters can be evaluated with any suitable reporter plasmid such as pcDNA3.1/V5-His/lacZ (Invitrogen
Catalog No. V810-20).
3. Mix the stock Lipofectamine 2000 gently before use,
and then dilute 2 ll in 50 ll Opti-MEM I (or other
medium without serum). Mix gently and let it stand
for 5 min at room temperature. (Note. The amount
of time diluted Lipofectamine 2000 is allowed to
stand prior complexation with DNA can aect transfection eciency. See Section 2.4).
4. Five minutes after dilution of Lipofectamine 2000,
combine the diluted DNA with the diluted lipid (total
volume is 100 ll). Mix gently and let it stand for
20 min at room temperature to allow the DNALipofectamine 2000 lipoplexes to form. The solution may
appear cloudy, but this will not inhibit transfection.
DNALipofectamine 2000 complexes are stable for
6 h at room temperature.
5. Add 100 ll of transfection complex to each well containing cells and medium. Mix gently by rocking the
plate back and forth.
6. Incubate the cells at 37 C in a humidied incubator
with 5% CO2 for 2448 h until they are ready to assay

Variations in cell density at the time of transfection

are a common source of variations in expression. Cells
should be counted and care should be taken to maintain
a standard seeding protocol. A cell density that is too
high can result in inhibition of cell growth and metabolism following transfection. Conversely, if cell density
is too low, recovery of the culture from the aects of
transfection can be poor. In general, cells should be
seeded to give a density of 9095% conuence to minimize the decrease in cell growth sometimes associated
with highly ecient transfection. The impact of cell
density is shown in Fig. 1. In this experiment, decreasing
numbers of 293 cells were seeded in a 24-well plate 18 h
before transfection with the plasmid pCMVSport6b-gal.
As done for all experiments reported here, 24 h from the
start of transfection cell lysates were prepared. Total
b-galactosidase activity was measured by ONPG assay
and is shown as nanograms of b-galactosidase per cm2
of well surface area. As would be expected, the total

Fig. 1. Eect of cell density on total reporter gene activity. 293-H cells
grown in 24 well plates were transfected with 0.8 lg of a b-galactosidase reporter plasmid and dierent volumes of Lipofectamine 2000, as
shown. b-Galactosidase activities, as ng b-galactosidase/cm2 well area,
were measured using an ONPG assay.

B. Dalby et al. / Methods 33 (2004) 95103

97

b-galactosidase expression is proportional to the starting

cell density. However, note also that as the cell density is
reduced the optimal amount of Lipofectamine 2000 is
also reduced, but not directly proportionate to the reduction in cell number.
2.3. Amount of DNA and DNA:Lipofectamine 2000 ratio
Lipofectamine 2000 is supplied as a 1 mg/ml solution.
In general, the mass ratio of DNA to Lipofectamine
2000 should be 1:2 to 1:3, although at higher DNA
amounts, a greater ratio can be employed. For example,
in a typical transfection at 9095% conuence in a 24well format, 0.81.0 lg plasmid DNA and 23 ll Lipofectamine 2000 can be used. The eect of DNA to
Lipofectamine 2000 ratio is shown in Fig. 2. When the
amount of plasmid DNA is held constant at either 0.2 or
0.8 lg, an optimal amount of Lipofectamine 2000 can be
seen, although with 0.2 lg DNA at least a 10-fold excess
of Lipofectamine 2000 is required. However, when
the recommended amount of DNA is used (0.8 lg) the
maximum total b-galactosidase activity is more than
twofold higher than with 0.2 lg DNA. Therefore even
when the DNA:Lipofectamine 2000 ratio is optimized,
sucient DNA must be used to allow high levels of
transgene expression.
2.4. Dilution time of Lipofectamine 2000 before adding
DNA
Prior to mixing with DNA, Lipofectamine 2000
should be diluted in Opti-MEM I. Depending on the
size of culture vessel being used, the Lipofectamine
2000 is diluted between 25- and 50-fold. As shown in
Fig. 3, the time between dilution of Lipofectamine 2000
and addition of DNA aects transfection eciency.
After dilution, Lipofectamine 2000 must be allowed to
incubate for a minimum of 5 min. Peak transfection

Fig. 3. Eect of Lipofectamine 2000 dilution time. COS7-L cells were

transfected with a b-galactosidase reporter plasmid as described in
Protocol 1. b-galactosidase activities, as ng b-galactosidase/cm2 well
area, were measured using an ONPG assay.

eciency is usually observed when the lipid is diluted

for 2030 min. Transfection eciency declines if the
lipid is not complexed with DNA within 6090 min of
dilution.
2.5. DNA:Lipofectamine 2000 complex formation
When diluted DNA and Lipofectamine 2000 are
mixed, liposomeDNA complexes are not formed immediately. To examine the optimal complexation time,
CHO-S cells were transfected with the b-galactosidase
reporter plasmid as described in Protocol 1.
As shown in Fig. 4, b-galactosidase expression is
signicantly improved as the time allowed for complexation increases, with optimal complex formation
achieved at about 30 min. Beyond 30 min complex formation time, the DNAlipid mix slowly decreases in
activity, as indicated by b-galactosidase expression.
However, the DNALipofectamine 2000 complexes are
relatively stable and continue to provide high levels of
expression 2 h after mixing, and even 6 h after mixing
(data not shown).
2.6. Time course of expression
To assess the early time course of transgene expression following transfection with Lipofectamine 2000,

Fig. 2. Eect of amount of DNA and DNA:Lipofectamine 2000 ratio.

293-H cells grown in 24 well plates were transfected with either 0.2 or
0.8 lg of a b-galactosidase reporter plasmid and dierent amounts of
Lipofectamine 2000, as shown. Mass ratios of DNA to Lipofectamine
2000 are shown for each DNA mass. b-galactosidase activities, as ng
b-galactosidase/cm2 well area, were measured using an ONPG assay.

Fig. 4. Eect of DNA:Lipofectamine 2000 mixing time. CHO-S cells

were transfected with a b-galactosidase reporter plasmid as described
in Protocol 1. b-Galactosidase activities, as ng b-galactosidase/cm2 well
area, were measured using an ONPG assay.

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B. Dalby et al. / Methods 33 (2004) 95103

293-H cells were transfected as described, cell lysates

were prepared at various time points, and b-galactosidase activity was measured. Transgene expression can be
detected as soon as 1 h from the start of transfection.
Expression continues to increase for at least 24 h from
the start of transfection (Fig. 5). Depending on the relative stability of the mRNA and protein expressed, and
the sensitivity of the assay system employed, this expression prole can vary signicantly. However, the
results obtained establish that plasmids can enter the
nucleus within 1 h of transfection. Peak transient expression is generally seen 2472 h following transfection,
depending as noted above, on the characteristics of the
gene being expressed.
2.7. Presence or absence of serum in culture medium
during transfection

Fig. 6. Eect of serum in culture medium during transfection. The cell

types shown were transfected with the b-galactosidase reporter plasmid
as described in Protocol 1. For each cell type, total b-galactosidase
activity in the presence of serum is taken as 100%. And compared with
relative activity in the absence of serum.

2.8. Cell culture conditions

One important consideration for the use of any
transfection reagent is the ability to function in the
presence of cell culture media supplemented with serum. This allows the reagent, if needed, to be
complexed with DNA and added directly to complete
cell culture medium. This eliminates the need to remove the complete medium before transfection and
to replace it afterwards. The transfection activity of
Lipofectamine 2000 in complete, serum containing
media was tested using several common cell lines.
Transfections were performed as described in Protocol 1, or by replacing the cell culture medium before
transfection with serum-free medium and then adding
back complete, serum-containing medium 45 h after
the start of transfection. For each of the cell lines
tested, there was less than 5% dierence in the
b-galactosidase activity between medium containing
serum and serum-free medium, indicating that serum
does not signicantly aect transfection eciency of
Lipofectamine 2000 (Fig. 6).

While the eect of cell culture conditions has not been

systematically examined in this study, the general health
of cells prior to transfection is known to be an important
source of variability from one transfection to another.
For example, cultures should not be maintained for
longer than 30 passages after thawing of a stock culture.
For optimal reproducibility, aliquots of cells of a low
passage number can be stored frozen and thawed as
needed. Excessive passaging is likely to detrimentally
aect transfection eciency (proportion of transfected
cells) as well as total transgene expression level from the
cell population as a whole. Also cells should be maintained in exponential growth and should never be allowed to become 100% conuent as this can reduce their
ability to resume exponential growth after passaging.

3. Transfection of siRNAs using Lipofectamine 2000

3.1. General considerations

Fig. 5. Time course of b-galactosidase reporter gene expression following transfection with Lipofectamine 2000. CHO-S cells were
transfected as described in Protocol 1. b-Galactosidase activities,
as ng b-galactosidase/cm2 well area, were measured using an
ONPG assay.

Lipofectamine 2000 has been used successfully to

transfect synthetic short interfering RNAs (siRNA) into
mammalian cells for RNA interference (RNAi) studies
[68]. A general protocol for the delivery of siRNAs
directed against a luciferase reporter gene and the lamin
A/C gene as described by Elbashir et al. [6] is presented.
A number of factors can inuence the degree to which
expression of a target gene is reduced in an RNAi experiment, including transfection eciency, transcription
rate of the gene of interest, mRNA and protein stability,
ecacy of the particular siRNA sequence chosen, and
the growth characteristics of the particular mammalian
cell line used. Hence, these factors should be considered
in the design of any RNAi experiment. Guidelines for
selection of siRNA sequences and preparation of siRNA
duplexes are given by Elbashir et al. [9].

B. Dalby et al. / Methods 33 (2004) 95103

3.2. Protocol 2: a general procedure for transfection of

siRNAs in a 24-well format
1. One day before transfection, plate cells in 0.5 ml of
growth medium (without antibiotics) so that cells will
be 3050% conuent at the time of transfection.
2. For each transfection sample, prepare siRNA:Lipofectamine 2000 complexes as follows: dilute the appropriate amount of siRNA in 50 ll Opti-MEM I
without serum (or other medium without serum).
Mix gently. Examples of the number of cells and
amounts of siRNA to use are given in Table 1.
3. Mix Lipofectamine 2000 gently before use and then
dilute the appropriate amount in 50 ll Opti-MEM I
(Invitrogen, Catalog No. 31985-062). Mix gently
and incubate for ve minutes at room temperature.
The considerations for the length of time diluted
Lipofectamine 2000 should be allowed to stand prior
to complexation with DNA are the same as described
in Sections 2.4 and 2.5.
4. After the 5-min incubation, combine the diluted
siRNA with the diluted Lipofectamine 2000 (total
volume is 100 ll). Mix gently and incubate for 20 min
at room temperature to allow the siRNA:Lipofectamine 2000 complexes to form.
5. Add the 100 ll of siRNA:Lipofectamine 2000 complexes to each well. Mix gently by rocking the plate
back and forth.
6. Incubate the cells at 37 C for 2472 h until they are
ready to assay for gene knockdown. It is generally
not necessary to remove the complexes or change
the medium; however, growth medium may be
replaced after 46 h without loss of transfection
activity.

99

expression. The sequence of each siRNA is shown in

Fig. 7A. Each siRNA has an additional 30 TdT overhang. siRNAs were obtained from Xeragon (www.
xeragon.com). Transfections were performed using
Protocol 3, except that with CHO-K1 cells only 10 pmol
siRNA was required to achieve knockdown. Luciferase
activity, relative to the activity in untransfected cells
(100%), was assayed 24 h from the start of transfection.
In each cell type, delivery of the luciferase siRNA resulted in greater than 80% knockdown of the luciferase
activity seen in untransfected cells. In contrast, delivery
of the nonspecic siRNA did not signicantly reduce
luciferase activity (Fig. 7B).
3.4. Knockdown of endogenous lamin A/C expression
In HeLa cells, reduction in the amount of the nuclear
envelope protein lamin A/C was demonstrated by introduction of a specic siRNA [6]. Knockdown of lamin
A/C protein levels, using the same siRNA, can also be
accomplished by transfection using Lipofectamine 2000.
Transfection was performed as described in Protocol 2
with the following modications: 6
104 HeLa cells

3.3. Knockdown of a luciferase reporter gene

To quantitate siRNA mediated gene knockdown, a
single copy of a luciferase reporter gene was stably integrated into 293, CHO, and BHK-21 cells using the
Flp-In system (Invitrogen). A 21 bp siRNA directed
against nucleotides 153173 of the Photinus pyralis luciferase (GL2 variant) open reading frame was used [6].
A 21 bp siRNA directed against GFP was used to control for nonspecic eects of siRNA delivery on gene

Table 1
Conditions for transfection of siRNAs using Lipofectamine 2000
Cell
line

Cell density
(cells/well)

Amount of
Lipofectamine
2000 (ll)

Amount
of siRNA
(pmol)

293
BHK
CHO-K1
A549

1
105
1.5
104
4
104
1.5
104

1
1
1
1

20
20
20
20

Fig. 7. (A) Sequences of siRNAs directed against luciferase and GFP

genes. (B) Knockdown of a luciferase activity in stably transfected cell
lines. Luciferase activity is measured relative to untransfected control
for each cell type. (C) Lamin A/C knockdown in HeLa cells. Lamin A/
C specic and GFP control nonspecic siRNAs were transfected into
HeLa cells using Protocol 2. Lamin A/C levels were compared by
Western blotting, using an antilamin A/C antibody. When cells are
transfected with the specic siRNA, lamin A/C protein cannot be
detected. In cells transfected with the nonspecic (GFP) siRNA, the
level is similar to that in untransfected cells.

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B. Dalby et al. / Methods 33 (2004) 95103

were seeded per well in DMEM 10% FBS, to give a

density of 90% at the time of transfection, and 60 pmol
lamin A/C siRNA (obtained from Dharmacon,
www.scbt.com) or a GFP siRNA (Xeragon) was used,
per well. Lamin A/C protein levels were evaluated by
Western blot two days from the end of transfection.
Cells were removed from the plate by using 0.25%
trypsin (Invitrogen Catalog No. 15050-065), washed in
PBS, and lysed in 40 ll of 1
LDS loading buer
(diluted from 4
, Invitrogen Catalog No. NP0007).
Samples were run on a 412% TrisBis NuPAGE
gel (Invitrogen Catalog No. NP0321), transferred to a
PVDF membrane, and blotted with an anti-lamin A/C
antibody (Santa Cruz Biotechnology, www.scbt.com).
Following detection of lamin A/C, the blots were
stripped and reprobed with an anti-actin antibody
(Santa Cruz Biotechnology). Chemiluminescent detection was accomplished using a Western Breeze kit (Invitrogen Catalog No. WB7104). As shown in Fig. 7C,
the level of lamin A/C protein in lysates from
mock transfected cells is similar to that in cells treated
with a nonspecic (GFP) siRNA. However, in cells
treated with the lamin A/C specic siRNA, lamin A/C
proteins cannot be detected. In each lysate, the level of
actin is similar showing equal protein loading in each
lane.

4. Transfection of primary neurons using Lipofectamine

2000
4.1. General considerations
The transfection of primary cells is often problematic
and very low transfection eciencies are common.
Neurons are an important class of primary cells where
improved transfection eciencies would facilitate study
of neurogenesis, neurophysiology, and pathology. The
protocol below gives transfection eciencies in rat hippocampal and cortical neurons of 2030% (Fig. 8), a
great improvement on transfection eciencies of less
than 3% previously reported. Ohki et al. [10] describe
this protocol and present additional data.
4.2. Protocol 3: culture of rat hippocampal and cortical
neurons and transfection using Lipofectamine 2000
1. Primary embryonic day 18 rat cortical or hippocampal tissue was obtained from BrainBits, (Springeld,
IL). Tissue is shipped in 2 ml vials of Hibernate-E
[11] supplemented with B27 [12] (Invitrogen Catalog
No. 17504-044) and used immediately upon arrival.
2. In preparation for dissociation, remove 1 ml of the
medium from the vial and place aside.

Fig. 8. (A) Embryonic day 18 (E18) cortical neurons (nontransfected control) xed and stained ve days after plating. (B) E18 cortical neurons
transfected with a b-galactosidase reporter plasmid using Lipofectamine 2000 4 days after plating, using Protocol 3). Cells were stained for b-galactosidase activity and photographed at 200
magnication 24 h later. In this experiment approximately 25% of the cells expressed b-galactosidase.
(C) E18 hippocampal neurons (nontransfected control) xed and stained 5 days after plating (200
magnication). (D) E18 hippocampal neurons
transfected with a b-galactosidase reporter plasmid using Lipofectamine 2000 4 days after plating. Cells were stained for b-galactosidase activity and
photographed at 200
magnication 24 h later. In this experiment, approximately 27% of the cells expressed b-galactosidase.

B. Dalby et al. / Methods 33 (2004) 95103

3. Using a sterile 9 in. Pasteur pipet, dissociate the tissue by repeated trituration (30 times) until the tissue
is homogeneously dispersed in the media.
4. Following dissociation, place previously removed
1 ml of media back into the vial.
5. Transfer the contents of the vial to a 15 ml tube and
centrifuge at 200g for 1 min.
6. Aspirate the supernatant and resuspend the pellet in
12 ml Neurobasal medium (Invitrogen Catalog No.
21103-049) with B27, 25 lM glutamic acid, and
0.5 mM L -glutamine [11]. Triturate the suspension
and place 0.5 ml of cell suspension in each well of
a 24-well plate. Plates are placed in a humidied incubator at 37 C, 5% CO2 . Prior to adding cells,
plates should be coated with 0.15 ml/cm2 poly-D -lysine (0.3 ml/well, 50 lg/ml, 135 kDa, Sigma Chemical, St. Louis, MO) solution. Between 9.0
104
and 5.9
105 cells can be seeded per well.
7. Three days after cell seeding, remove 0.25 ml of
the media and replace with fresh Neurobasal medium with B27, 0.5 mM L -glutamine but without
glutamate. Cells are incubated 24 h prior to transfection.
8. Dilute 8 ll Lipofectamine 2000 in 100 ll OptiMEM I
per well and incubate at room temperature for
5 min. Combine 100 ll OptiMEM I with 12 lg
DNA and incubate at room temperature for 5 min.
Mix the diluted DNA and Lipofectamine 2000 and
incubate at room temperature for 20 min.
9. Replace the culture medium in each well with 400 ll
fresh Neurobasal/B27/L -glutamine medium. Add
100 ll OptiMEM I containing DNA/Lipofectamine
2000 transfection complexes to each well. Transgene
expression can be evaluated 24 h from the start of
transfection.
10. If cells are transfected with a b-galactosidase reporter plasmid such as pcDNA3.1/V5-His/lacZ (Invitrogen Catalog No. V810-20) transfection
eciency can be evaluated by the proportion of cells
that stain positive for b-galactosidase activity, using
the Invitrogen b-gal staining kit (Catalog No.
K1465-01).

5. High throughput (HTP) transfection

5.1. General considerations
The availability of cloned gene collections together
with the development of high throughput cell based
assays has led to a need for simplied transfection
procedures adapted to 96-well or 384-well formats.
Protocols have been developed that use Lipofectamine 2000. Depending on assay requirements, transfection of a single plasmid may be performed using a
large volume of cells in suspension, which are then

101

distributed among a number of individual wells for

subsequent treatment and assay. Alternatively, there
may be the need to transfect a large number of
dierent cloned genes such that each well of transfected cells expresses a dierent transgene. The protocols given below can be adapted for either HTP
transfection requirement. For HTP assays where a
cell line of human origin is preferred, the GripTite
293-MSR line (Invitrogen Catalog No. R79507) is
recommended. GripTite 293-MSR cells are stably
transfected with the Macrophage Scavenger receptor
(MSR) to give improved adhesion to standard tissue
culture treated plastic surfaces, eliminating the need
for poly-D -lysine coated plates [13]. GripTite 293MSR cells can be used in conjunction with automated cell washing and assay procedures that cannot
be performed easily on 293 cells lacking the MSR,
because many cells would be lost due to poor cell
adhesion.
5.2. Protocol 4: transfection of single or multiple genes in
a 96-well format
1. Seed cells in 96-well plates to give a density of approximately 90% at the time of transfection Use
4
104 GripTite 293-MSR cells per well in 100 ll
DMEM (Invitrogen Catalog No. 11885-084 plus
10% FBS (Invitrogen Catalog No. 11885-084).
2. For each well of cells, dilute 0.20.3 lg DNA in 25 ll
OptiMEM I reduced serum medium and dilute 0.5 ll
Lipofectamine 2000 in 25 ll OptiMEM I. Incubate
diluted Lipofectamine 2000 at room temperature
for 5 min. For multiple genes, one Lipofectamine
2000:DNA mixture is prepared for each gene and
scaled for the number of wells used allowing for replicates. For a single DNA, the dilution volumes of
Lipofectamine 2000 and the DNA can be increased
using the proportions given above, while maintaining
a diluted DNA volume of 25 ll per well and the same
diluted Lipofectamine 2000 volume.
3. Mix diluted Lipofectamine 2000 and DNA. Incubate
at room temperature for 2030 min and then add
50 ll of the transfection complexes to each well. Assay for transgene expression 24 h from the start of
transfection.
5.3. Protocol 5: an alternative rapid cell plating and
transfection protocol
1. Prepare Lipofectamine 2000:DNA complexes as described in Protocol 4, steps 2 and 3.
2. Add 50 ll diluted Lipofectamine 2000:DNA to each
empty well of a 96-well plate.
3. Immediately add 6
104 GripTite 293-MSR cells to
each well. Transfection occurs while cells are attaching to the well.

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B. Dalby et al. / Methods 33 (2004) 95103

4. Assay for transgene expression 24 h from the end of

transfection.
5.4. Protocol 6: rapid transfection of a single DNA for
high throughput assay
1. Transfection of a single DNA can be performed with
the cells in suspension, prior to plating: 6
105 GripTite 293-MSR cells are diluted per ml of DMEM/10%
FBS medium.
2. For each milliliter of cells, dilute 10 ll Lipofectamine
2000 in 500 ll OptiMEM I medium and incubate at
room temperature for 5 min. Dilute 23 lg DNA in
500 ll OptiMEM I medium. Mix and incubate at
room temperature for 20 min.
3. Mix cells and diluted Lipofectamine 2000:DNA
transfection complexes. Incubate at 37 C, 5% CO2 ,
with humidied air for 3 h. Add 100 ll of complete
mixture to each well of a 96-well plate.
Transfection eciencies and well-to-well variation
were compared for the protocols given above. GripTite
293-MSR cells were transfected using 0.2 or 0.3 lg
pDNA3.1/V5-His/lacZ DNA per well and either 0.5 or

1 ll Lipofectamine 2000 per well. Lipofectamine

2000:DNA complexes were prepared as above and then
applied to cells in three ways; complexes were added to
cells that had already been allowed to attach to the
plate (Fig. 9A, upper two rows of stained cells, Protocol 4, steps 13). Alternatively cells together with
Lipofectamine 2000:DNA complexes were added to
wells so that transfection could occur as cells were
attaching to the plate (Fig. 9B middle two rows of
stained cells, Protocol 5). In the third method, Lipofectamine 2000:DNA complexes were mixed with cells
in suspension and the mixture was then added to the
empty wells (Fig. 9C, bottom two rows of stained cells,
Protocol 6, above). Twenty-four hours from the start
of transfection, all wells were stained for b-galactosidase activity using the Invitrogen b-gal Staining kit
(K1465-01).
All wells that were transfected using 0.5 ll Lipofectamine 2000 show similar b-galactosidase staining intensities. When a twofold excess of Lipofectamine 2000
is used (1 ll/well), fewer stained cells remain. There is no
detectable dierence between wells treated with either
0.2 or 0.3 lg DNA per well.

Fig. 9. (a) Transfection of GripTite293 MSR cells with a b-galactosidase reporter plasmid using Lipofectamine 2000. (A) Lipofectamine 2000:DNA
complexes were added to cells that had already been allowed to attach to the plate (B). Lipofectamine 2000:DNA complexes were added to wells so
that transfection could occur as cells were attaching to the plate. (C). Lipofectamine 2000:DNA complexes were mixed with cells in suspension and
the mixture was then added to the empty wells. For each method, either 0.2 or 0.3 lg DNA per well was used and either 0.5 or 1 ll Lipofectamine
2000. All wells were stained for b-galactosidase reporter activity 24 h after transfection. (b) Total b-galactosidase activities corresponding to
transfection protocols A, B or C above. b-Galactosidase activities, as ng b-galactosidase/cm2 well area, were measured using an ONPG assay.

B. Dalby et al. / Methods 33 (2004) 95103

Acknowledgments
The author thanks Shelley Bennett, Krista Evans,
Sheryll Mangahas, Jean Pierre Pichet, and Kevin
Schierli for technical support.

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