Lipofectamine® 2000 CD Reagent

Introduction

Lipofectamine®  2000 CD is a proprietary animal origin-free formulation for the transfection of nucleic acids into eukaryotic cells. A Drug Master File (DMF) has been submitted to the FDA. Contact Technical Service or your local Sales Representative for permission to cross-reference the DMF. Using Lipofectamine® 2000 CD for transfection provides the following advantages:

Highest transfection efficiency in many cell types and formats. Refer to the Cell Lines database at www.invitrogen.com for a list of cell types transfected. he animal origin-free formulation ensures that mammalian cell culture and bioproduction processes are free of animal-derived materials. DNA-Lipofectamine® 2000 CD complexes can be added directly to cells in culture medium. It is not necessary to remove complexes or change/add medium after transfection, but complexes may be removed after 4-6 hours.

Important Guidelines for Transfection

  1. Culture cells in serum-free media that are free of animal-derived components. Test serum-free media for compatibility with Lipofectamine® 2000 CD since some serum-free formulations (e.g. CD 293, 293 SFM II, CD Hybridoma) may inhibit cationic lipid-mediated transfection.
  2. For consistent animal origin-free transfection, use OptiPro™ SFM (Cat. No. 12309-019) to dilute DNA and Lipofectamine® 2000 CD before complexing.
  3. Transfect cells at high cell density: For adherent cells, 90-95% confluence at the time of transfection is recommended for high efficiency, high expression levels, and to minimize cytotoxicity. For suspension cultures, transfect cells at a density of 0.8-1.1 x 106 cells/ml. Optimization may be necessary. Maintain the same seeding conditions between experiments.
  4. Do not add antibiotics to media during transfection as this causes cell death.
  5. Do not add Pluronic® or charged media extracts (e.g. dextran sulfate) to media during transfection as these reagents can inhibit transfection
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Ordering Information

Sku Name Size Price Qty
12566014 Lipofectamine® 2000 CD Transfection Reagent 1 mL USD 544.00

Transfecting Adherent Cells

Use the following procedure to transfect mammalian cells in a 24-well format. For other formats, see Scaling Up or Down Transfections. Use the recommended Lipofectamine® 2000 CD amount as a starting point, then optimize conditions for your cell line and serum-free medium, as needed. All amounts and volumes are given on a per well basis.

  1. One day before transfection, plate 0.5-2 x 105 cells in 500 μl of growth medium without antibiotics so that they will be 90-95% confluent at the time of transfection.
  2. For each transfection sample, prepare complexes as follows

    • a. Dilute DNA in 50 μl of OptiPro™ SFM. Mix gently.
    • b. Mix Lipofectamine® 2000 CD gently before use, then dilute the appropriate amount in 50 μl of OptiPro™ SFM. Incubate for 5 minutes at room temperature. Note: Combine diluted Lipofectamine® 2000 CD with diluted DNA within 30 minutes.
    • c. After 5 minute incubation, combine the diluted DNA with diluted Lipofectamine®  2000 CD (total volume = 100 μl). Mix gently and incubate for 20 minutes at room temperature (solution may appear cloudy). Note: Complexes are stable for 6 hours at room temperature.
  3. Add the 100 μl of complexes to a well containing cells and medium. Mix gently by rocking the plate back and forth.
  4. Incubate cells at 37°C in a CO2 incubator for 18-48 hours prior to testing for transgene expression. It is not necessary to change the medium, but medium may be replaced after 4-6 hours.
  5. For stable cell lines: Passage cells at a 1:10 (or higher dilution) into fresh growth medium 24 hours after transfection. Add selective medium the following day. 
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Scaling Up or Down Transfections

To transfect cells in different tissue culture formats, vary the amounts of Lipofectamine® 2000 CD, DNA, cells, and medium used in proportion to the relative surface area, as shown in the table. With automated, high-throughput systems, a complexing volume of 50 μl is recommended for transfections in 96- well plates.

Culture vessel Surf. area
per well
(cm2)
Relative
surf. area
vs. 24-well
Vol. of
plating
medium
DNA (μg) in
media vol. (μl)
Lipofectamine®
2000 CD (μl) in
media vol. (μl)
96-well0.30.2100 μl0.2 μg in 25 μl0.5 μl in 25 μl
24-well21500 μl0.8 μg in 50 μl2.0 μl in 50 μl
12-well421 ml1.6 μg in 100 μl4.0 μl in 100 μl
35-mm105 4.0 μg in 250 μl10 μl in 250 μl
6-well1052 ml4.0 μg in 250 μl10 μl in 250 μl
60-mm20105 ml8.0 μg in 0.5 ml20 μl in 0.5 ml
10-cm603015 ml24 μg in 1.5 ml60 μl in 1.5 ml


Note: Surface areas are determined from actual measurements of tissue culture vessels, and may vary depending on the manufacturer.

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Transfecting Cells in Suspension

Use the following procedure to transfect suspension cells at any scale. Before beginning, make sure that cells are healthy and > 90% viable.

  1. On the day of transfection, prepare a single cell suspension from stock cells at < 3 x 106 cells/ml. Seed cells at a density of 0.8-1.1 x 106 cells/ml in growth media without antibiotics.
  2. For each transfection sample, prepare complexes as in Step 2, using the following reagent amounts and volumes for every ml of cells transfected:

    • Dilute 0.5-1.5 μg DNA in 34 μl of OptiPro™ SFM
    • Dilute 1-10 μl of Lipofectamine® 2000 CD in 34 μl of OptiPro™ SFM
  3. Add the complexes to the flask containing cells and media.
  4. Incubate the cells at 37°C in a CO2 incubator on an orbital shaker rotating at 125 rpm for 24-96 hours prior to testing for transgene expression.


Optimizing Transfection

To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying cell density and Lipofectamine® 2000 CD concentrations.

Quality Control

Lipofectamine® 2000 CD 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.

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12566014.pps      20-Jul-2004