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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
96
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.
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
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.
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98
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.
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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
100
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.
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).
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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.
Acknowledgments
The author thanks Shelley Bennett, Krista Evans,
Sheryll Mangahas, Jean Pierre Pichet, and Kevin
Schierli for technical support.
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