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Lipofectamineâ„? 2000 Transfection Reagent
TABLE OF CONTENTS
PRODUCT DESCRIPTION
SHIPPING CONDITIONS
STORAGE CONDITIONS
STABILITY
QC SPECIFICATIONS
PROTOCOL & APPLICATION NOTES
Relative Surface Areas Of Tissue Culture Vessels
Surface Areas Of Tissue Culture Vessels
Tubes Recommended For Use With Lipofectamineâ„? 2000
Optimizing Plasmid DNA Transfections With Lipofectamineâ„? 2000
Transfection Protocols
Co-Transfection Of Sirna And Plasmid DNA
Transfection Of Fluorescently Labeled Oligos With Lipofectamineâ„? 2000
Transfection Of Cells In 96-Well Plates
Transient Transfection Of Suspension Cells
ALTERNATE PRODUCTS & COMPATIBILITY
PRODUCT DOCUMENTATION
REFERENCES
PRODUCT NAME & CATALOG NUMBERS
ASSOCIATED PRODUCTS
RELATED TECHNICAL SUPPORT NOTES
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PRODUCT DESCRIPTION
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Lipofectamineâ„? 2000 Transfection Reagent is a proprietary formulation for the transfection of nucleic acids (DNA and RNA)
into eukaryotic cells and provides the following advantages:
Highest transfection efficiency in many cell types and formats (e.g. 96-well). Refer to the Cell Lines Database for a
list of cell types successfully transfected. Detailed in-house transfection protocols are also available at this site where
available.
DNA-Lipofectamineâ„? 2000 complexes can be added directly to cells in culture medium, in the presence or absence
of serum.
Lipofectamineâ„? 2000 may be used in the following applications:
Transient and stable transfection of adherent and suspension cells
High throughput transfections
Delivery of Stealth RNAi and siRNA into cells For information on transfecting mammalian cells with short
interfering RNAs (siRNA) for use in RNA interference (RNAi) studies, visit our RNAi Central web page at
www.invitrogen.com\rnai. Cell line-specific protocols are available under the Protocols tab.
Lipofectamineâ„? 2000 gives superior transfection efficiency for the following cell lines:
293F 293H BE(2)C (w/o serum)
CHO-K1 CHO-S (adherent) COS-1 (w/o serum)
COS7-L Primary Human Fibroblasts (w/o serum)
HT-29 (w/o serum) HT-1080 MDCK
MRC-5 (w/o serum) PC12 SK-BR3
Vero HepG2 NIH 3T3
Lipofectamineâ„? 2000 CD is a 100% synthetic version of Lipofectamineâ„? 2000. Use it in the same way as Lipofectamineâ„?
2000, but be sure to use animal origin-free reagents.
SHIPPING CONDITIONS
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This kit is shipped on wet ice.
STORAGE CONDITIONS
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Lipofectamineâ„? 2000 Transfection Reagent should be stored at 40 C.
STABILITY
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The stability of Lipofectamineâ„? 2000 Transfection Reagent is guaranteed for 6 months when it has been stored as
recommended.
Lipofectamineâ„? 2000 Reagent should not be frozen.
QC SPECIFICATIONS
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Lipofectamineâ„? 2000 is tested for the absence of microbial contamination using blood agar plates, Sabaraud dextrose agar plates,
and fluid thioglycolate medium, and functionally by transfection of CHO-K1 cells with a reporter plasmid.
PROTOCOL AND APPLICATION NOTES
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General protocol notes
Surface Areas Of Tissue Culture Vessels
Tubes Recommended For Use With Lipofectamineâ„? 2000
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Optimizing Plasmid DNA Transfections With Lipofectamineâ„? 2000
Transfection Protocols
Co-Transfection Of Sirna And Plasmid DNA
Transfection Of Fluorescently Labeled Oligos With Lipofectamineâ„? 2000
Transfection Of Cells In 96-Well Plates
Transient Transfection Of Suspension Cells
General protocol notes
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(back to Protocol and Application Notes)
It is not necessary to remove complexes or change/add medium after transfection, but complexes may be removed after 4-
6 hours.
DMEM or RPMI 1940 can be used instead of Opti-MEM when making Lipofectamine� 2000 � DNA complexes.
However, the efficiency of complex formation may not be as high as with Opti-MEM. Cells in PBS can be transfected
using Lipofectamineâ„? 2000.
As long as the cells are healthy in the PBS, the transfection is likely to work.
For a general plasmid DNA transfection protocol, please refer to the product insert:
http://www.invitrogen.com/content/sfs/manuals/Lipofectamineâ„?2000_man.pdf
A general protocol for transfecting Stealth RNAi or siRNA into mammalian cells can be found at the following site:
http://www.invitrogen.com/content/sfs/manuals/stealth_sirna_tsf_lf2k_man.pdf
Surface Areas of Tissue Culture Vessels
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(back to Protocol and Application Notes)
Culture Vessel 96- 48-well 24-well 12-well 6-well 35-mm 60-mm 100-mm 150-mm T25 T75
well
Surface Area (cm2) 0.3 0.7 2 4 10 10 20 60 140 25 75
Ratio to 24-well plate 0.2 0.4 1 2 5 5 10 30 70 12.5 37.5
Tubes recommended for use with Lipofectamineâ„? 2000
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It is best to use polypropylene tubes when pre-mixing Lipofectamine 2000 â„? and DNA. Polystyrene may not work as well.
Optimizing plasmid DNA transfections with Lipofectamineâ„? 2000
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The conditions that could be optimized include Lipofectamineâ„? 2000 amount, DNA concentration, and cell number. Keeping two
variables constant, vary the third.
For example: to optimize the amount of Lipofectamineâ„? 2000 for transfection in a 24-well plate, start with cells at >90%
confluency and use a fixed amount of DNA (0.8-1.2 µg). With cell number and DNA concentration held constant, vary the
amount of Lipofectamine� 2000 to determine the optimal concentration (usually 1.5-3 µl). In the same way, the cell number
and amount of DNA can also be optimized.
It is recommended to use a range of 0.5 to 5 µl of Lipofectamine� 2000 per µg of DNA. It is possible to minimize the effect
of transfection on cell growth and viability by increasing the number of cells plated per well or by decreasing either
Lipofectamineâ„? 2000 amount or DNA concentration. With careful optimization, this can be achieved with little impact on
the level of transgene expression.
Transfection efficiency is typically measured as the percentage of cells translating and accumulating the protein of interest for
detection in the total population. If the levels of translation or protein accumulation are low, a lower transfection efficiency
may be obtained. A transfection control such as our BLOCK-iT Fluorescent Oligo (catalog # 2013) is a more accurate
measure of the efficiency of DNA delivery since its detection is independent of expression in the cell.
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The following citation discusses the effect of variables such as cell density, liposome and DNA concentrations, liposome-
DNA complexing time, and media components (serum and antibiotics) on transfection with Lipofectamineâ„? 2000. In
addition, it also looks at high throughput transfections, siRNA transfections, and transfection of primary neurons.
Advanced transfection with Lipofectamineâ„? 2000 reagent: primary neurons, siRNA, and high-throughput applications -
Methods, Volume 33, Issue 2, June 2004, Pages 95-103 Brian Dalby, Sharon Cates, Adam Harris, Elise C. Ohki, Mary L.
Tilkins, Paul J. Price and Valentina C. Ciccarone
Though most cells transfect well in the presence or absence of serum, there are a few such as HeLa cells and Normal Human
Fibroblasts that give better transfection efficiency in the absence of serum.
Cell lines successfully transfected with Lipofectamineâ„? 2000:
293F 293H, 293 BE(2)C (w/o serum)
CHO-K1 CHO-S (adherent) CHO-S (suspension in CD CHO media)
COS-1 (w/o serum) COS7-L(w/o serum) Primary Human Fibroblasts
HT-29 (w/o serum) HT-1080 MDCK
MRC-5 (w/o serum) PC12 SK-BR3
Vero CHO CHO-DG44
MCF7 MDA-MB-361 HCT 116
H1299 RKO Hep3B, HepG2
HeLa Rzneo HOS
C3H/10T1/2 NIH3T3 Jurkat
K562 HUVECS LoVo
A549
Some cell lines for which transfection protocols are available (at the cell lines database)
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(back to Protocol and Application Notes)
Cell type Transfection Cells per well Lipofectamineâ„? 2000
µl per well in
Efficiency (%) (24-well plate)
24-well plate
2 x 105
293H 99 2
2 x 105
293F 99 2
2 x 105
BE(2)C 77 2.5
1.0 x 105
BHK21 - 3.0
1.2 x 105
CHO-K1 - 2.5
1.5 x 105
CHO-S(adherent) 96 2.5
8 x 104
Cos 1 - 3.0
8 x 104
COS7L 99 2.5
8 x 104
CV-1 70 1.5
8 x 104
HeLa 94 1.5
1.5 x 105
HT-29 - 3.0
8 x 104
HT1080 81 1.5
8.0 x 104
HUVEC <2% 2.0
6 x 104
MDCK 43 4
1.5 x 105
MRC-5 Not measured 2.5
1X106 (6-well)
Murine Embryonic Stem Cells, D3 (6- 75 8-12(6-well)
well plates)
1.5 x 105
NIH3T3 Not measured 2
2.5 X 105
PC12 85 2
8 x 104
Primary Human Fibroblasts 48 2
Primary Human Keratinocytes - 8 x 104 2
RKO (field test) (6-well plates) 40 - 60 See protocol See protocol
1.5 x 105
SKBR3 49 2
8 x 104
Vero 86 2
1.25 x 105
Rat Hepatocytes 50 1.5
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2 x 105
Rat E18 Cortical Neurons 20-25 4
Co-transfection of siRNA and plasmid DNA
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Plasmid and siRNA co-transfection are possible. Co-transfections have been tested with Lipofectamineâ„? 2000 in
GripTiteâ„? cells (293 derived cells) plated at 1.8 x 105 cells/well in a 24-well format (0.5ml medium, no antibiotics).
200ng of two different reporter plasmids were co-transfected with 10pmol of siRNA following the standard
Lipofectamineâ„? 2000 protocol, with 2ul of Lipofectamineâ„? 2000 per well. The total volume of the transfection mixes
was 100ul, and it was added to the medium already in the wells.
Transfection of fluorescently labeled oligos with Lipofectamineâ„? 2000
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Fluorescently labeled oligos that depend on hairpin structures for quenching (like LUX primers) may fluoresce upon mixing with
Lipofectamineâ„? 2000. In such cases Oligofectamine is suggested. Oligofectamine is very different chemically, and therefore not
expected to exhibit the same strength of interaction.
Transfection of cells in 96-well plates (also see Focus 21.3 page58)
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(back to Protocol and Application Notes)
Use the 24-well plate protocol with the following modifications:
Plate 2-6 x 104 cells per well in 100 µl of the appropriate complete growth medium without antibiotics and with serum if
cells are normally cultured in the presence of serum.
For each well of cells, dilute 240 to 320 ng of DNA into 25 µl medium without serum (e.g., OptiMEM® I Medium) in
96-well, sterile micro titer plates.
For each well of cells, dilute 0.8-1 µl of Lipofectamine� 2000 into 25 µl OptiMEM® Medium and incubate for 5 min at
room temperature. Once the Lipofectamineâ„? 2000 is diluted, combine it with the DNA within 30 min. Longer
incubation times may result in decreased activity. This dilution can be prepared in bulk for multiple wells.
Add 25 µl of the diluted Lipofectamine� 2000 (from step 3) to each well containing diluted DNA (from step 2), mix
gently, and incubate at room temperature for 20 min to allow DNA- Lipofectamineâ„? 2000 complexes to form.
Add the DNA- Lipofectamine� 2000 complexes (50 µl) directly to each well of the plates containing cells and mix
gently.
Optimal transfection conditions for transfections in 96-well plates:
Cell Line Seeding Density DNA per Well Lipofectamineâ„? 2000
(Cells per well) (µl)
1 µl
2 x 104
CHO-S 240 ng
1 µl
2.5 x 104
COS-7L 320 ng
1 µl
5 x 104
293H, 293F 320 ng
Alternate rapid protocol for 96-well transfections without pre-plating cells (also see Focus 21.3, page58):
This protocol is designed as a rapid alternative that does not require plating cells the day before transfection. Instead, a
suspension of cells is added directly to complexes prepared in 96-well plates. This protocol has been used successfully
with the cells and conditions outlined below. Use poly-lysine coated plates (D or L) for best results.
Dilute ~320 ng of each DNA to be tested into 25 µl medium without serum (e.g., OptiMEM® I Medium). Prepare the
dilutions directly in 96-well cell culture plates.
For each well, dilute 0.4-0.8 µl of Lipofectamine� 2000 into 25 µl OptiMEM® I Medium and incubate for 5 min at
room temperature. Prepare this dilution in bulk for multiple wells. Once the Lipofectamineâ„? 2000 is diluted, combine it
with the DNA within 30 min. Longer incubation times may result in decreased activity.
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Add 25 µl of the diluted Lipofectamine� 2000 (from step 2) to each well containing diluted DNA (from step 1), mix
gently, and incubate at room temperature for 20 min to allow DNA-Lipofectamineâ„? 2000 complexes to form.
Prepare a cell suspension so that the appropriate number of cells per well is contained in 100 µl of growth medium. Use
approximately twice the cell density, depending on cell type, than with the standard protocol.
Add 100 µl of the cell suspension (from step 4) to each of the wells containing the DNA-Lipofectamine� 2000
complexes (from step 3) and mix gently.
Incubate at 37oC in a CO2 incubator until ready to assay (24-48 h post transfection). It is not necessary to remove the
complexes or change the medium. Cells will adhere as usual in the presence of the complexes.
Transfection conditions for rapid 96-well protocol:
Cell Line Cells per well DNA per well Lipofectamineâ„? 2000
(100 µl suspension) per well
0.8 µl
5 x 104
CHO-S 320 ng
0.6-0.8 µl
6 x 104
COS-7L 320 ng
0.4 µl
1.2 x 105
293H, 293F 320 ng
Transient transfection of suspension cells
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The following protocol was optimized with Jurkat and K562 cells, but can be used as a guideline for other types of suspension
lines.
For each transfection, add a cell suspension containing 4-8 x 105 cells in 500 µl of growth medium with serum but
without antibiotics, to a well of a 24 well plate. For transfection of larger number of cells, scale up all the reagents (cells,
media, DNA, Lipofectamineâ„? 2000 and plate size) proportionately to the number of cells transfected.
For each well, dilute 0.8 - 1.2 µg of DNA into 50 µl of medium without serum (e.g., Opti-MEM). This can be prepared in
bulk for multiple wells.
For each well, dilute ~2 µl of Lipofectamine� 2000 into 50 µl OptiMEM® I Medium and incubate for 5 min at room
temperature. Once the Lipofectamineâ„? 2000 is diluted, combine it with the DNA within 30 min. Longer incubation
times may result in decreased activity. This dilution can be prepared in bulk for multiple wells.
Combine the diluted DNA from step 2 with the diluted Lipofectamineâ„? 2000 from step 3. Incubate at room temperature
for 20 min to allow DNA-Lipofectamineâ„?2000 complexes to form.
Add the DNA-Lipofectamine� 2000 complexes from step 4 (100 µl) directly to each well containing cells (from step 1)
and mix gently by rocking the plate back and forth.
Incubate for 4 h at 37oC in a CO2 incubator.
In Jurkat cells, addition of PHA-L (Phytohemagglutinin L) and PMA (phorbol myristate acetate) at final concentrations of
1 µg/ml and 50 ng/ml, respectively, enhances CMV promoter activity and gene expression. In K562 cells, PMA alone is
sufficient to enhance promoter activity. PMA and PHA are added after the 4-h incubation.
Assay the cells at 24-48 h post-transfection for the appropriate activity. It is not necessary to remove the complexes or
change the medium.
Note: Jurkat cells are difficult to transfect and have low expression following transfection.
The use of PHA-L and PMA did not affect the expression level of a beta-gal reporter (by ONPG assay) in either Jurkat cells or
K562 cells in our hands. They are widely used in transfecting these cell types, and their use is most likely historical. Their
effectiveness in transfections with currently available lipid reagents has probably not been tested before.
PRODUCT DOCUMENTATION
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Brochures Cell Lines Citations
COA FAQ Licensing
Manuals MSDS
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REFERENCES
(back to Table of Content)
Krista Evans, et al..PGreen Lantern-1, A Superior Green Fluorescent Protein Mammalian Cell Transfection Reporter,
Focus 18(2): 40.
Valentina Ciccarone, et al. Lipofectamineâ„? 2000 Reagent for Rapid, Efficient Transfection of Eukaryotic Cells - Focus
21(2):54.
Jean-Pierre Pichet and Valentina Ciccarone. Transfection of Mammalian Cells in 96-Well Plates with Lipofectamineâ„?
2000 Reagent. Focus 21(3):58.
Cationic Lipid Reagent Selection. Focus 21(3):61.
Linda Roy, et al. High Transfection Efficiency of Cloned Cell Lines. Focus 21(3):62.
Achieve the highest transfection efficiencies and higher expression levels (with Lipofectamineâ„? 2000). Expressions
8(3):18.
PRODUCT NAME AND CATALOG NUMBERS
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Name Size Part Number Catalog Nnumber
Lipofectamineâ„? 2000 Reagent 0.75 ml 52758 11668-027
(11668027)
Lipofectamineâ„? 2000 Reagent 1.5 ml 52887 11668-019
(11668019)
Lipofectamineâ„? 2000 CD Reagent 1.0 ml 52888 12566-014
(12566014)
ASSOCIATED PRODUCTS
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OPTI-MEM I Reduced Serum Medium (catalog # 31985-062)
Antibiotics
GIBCO® Cell Culture Products
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