Lipofectamine® Reagent for the transfection of DNA into eukaryotic cells

Introduction

Description

Lipofectamine® Reagent is suitable for the transfection of DNA into eukaryotic cells (1), and is a 3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,Ndimethyl- 1-propanaminium trifluoroacetate (DOSPA) and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) in membrane-filtered water. Transfection activity can be enhanced by using Plus™ Reagent (Cat. No. 11514-015)(2) to pre-complex the DNA. Refer to the Cell Lines database at www.invitrogen.com for protocols to transfect various cell types.

Important Guidelines for Transfection

  1. Prepare complexes using the amount of DNA and Lipofectamine® recommended below. Optimization may be necessary. Note: We recommend using Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) to dilute Lipofectamine® and DNA before complexing.

  2. Transfect cells at high cell density: 50-80% confluence at the time of transfection is recommended for high efficiency, high expression levels, and to minimize cytotoxicity. Optimization may be necessary. Maintain the same seeding conditions between experiments.

  3. Do not add antibiotics to media during transfection as this causes cell death.

  4. Test serum-free media for compatibility with Lipofectamine® since some serum-free formulations (e.g. CD 293, 293 SFM II, and VP-SFM) may inhibit cationic lipid-mediated transfection.


This product is distributed for laboratory research only. CAUTION: Not for diagnostic use. The safety and efficacy of this product in diagnostic or other clinical uses has not been established.

. TOP

Transfection Procedure

Use the following procedure to transfect adherent mammalian cells in a 24- well format. For other formats, see Scaling Up Transfections. All amounts and volumes are given on a per well basis.

  1. One day before transfection, plate 2-6 x 104 cells in 500 μl of growth medium (with the usual amount of serum) without antibiotics so that cells will be 50-80% confluent at the time of transfection.

  2. For each transfection sample, prepare complexes as follows:

    • a. Dilute 0.2-0.4 μg DNA in 25 μl of Opti-MEM® I Reduced Serum Medium (or other medium) without serum. Mix gently.
    • b. Mix Lipofectamine® gently before use, then dilute 0.5-5 μl in 25 μl of Opti-MEM® I Medium (or other medium) without serum. Mix gently.
    • c. Combine the diluted DNA with diluted Lipofectamine® (total volume = 50 μl). Mix gently and incubate for 15-45 minutes at room temperature (solution may appear cloudy). Note: Complexes are stable for 6 hours at room temperature.
    • d. For each transfection, add 0.15 ml of Opti-MEM® I Medium to the tube containing the complexes (total volume = 200 μl). Mix gently.

  3. Remove the growth medium from cells and replace with 0.2 ml of growth medium without serum. Add the 0.2 ml of diluted complexes (from Step 2d) to each well. Mix gently by rocking the plate.

  4. Incubate cells at 37°C in a CO2 incubator for 2-24 hours. We recommend starting with 5 hours.

  5. Add 0.4 ml of growth medium containing 2X the normal concentration of serum without removing the transfection mixture. Note: If toxicity is observed after transfection, replace medium with fresh, complete medium (with normal amount of serum).

  6. For transient transfection: Test for transgene activity 24-72 hours posttransfection as appropriate for your cell type and expression vector. For stable transfection: Passage cells at a 1:10 dilution into selective medium 72 hours post-transfection.
TOP

Scaling Up Transfections

To transfect cells in different tissue culture formats, vary the amounts of Lipofectamine®, DNA, cells, and medium used in proportion to the relative surface area, as shown in the table.

Culture vessel Relative surf. area vs. 24-well Vol. of plating medium DNA (μg) in media vol. (μl) Lipofectamine® (μl) in media vol. (μl) Transfection medium vol.
24-well1500 μl0.2-0.4 μg in 25 μl0.5-5 μl in 25 μl0.4 ml
35-mm52 ml1-2 μg in 100 μl2-25 μl in 100 μl0.8 ml
60-mm105 ml3-6 μg in 300 μl6-75 μl in 300 μl2.4 ml
10-cm3015 ml8-16 μg in 800 μl16-200 μl in 800 μl6.4 ml

 

TOP

Enhancing Transfections with Plus™ Reagent

Use this procedure to enhance transfections with Plus™ Reagent in a 24-well format. For other formats, vary the amounts of reagents used in proportion to the relative surface area (see table above).

  1. Perform Step 1 of the Transfection Procedure.

  2. For each transfection sample, prepare complexes as follows:

    • a. Dilute 0.4 μg DNA in 25 μl medium without serum (e.g. D-MEM).
    • b. Mix Plus™ Reagent before use, then add 4 μl to diluted DNA. Mix again, and incubate at room temperature for 15 minutes.
    • c. Dilute 1 μl Lipofectamine® in 25 μl medium without serum; mix.
    • d. Combine pre-complexed DNA (Step 2b) and diluted Lipofectamine® (Step 2c); mix and incubate for 15 minute  at room temperature.

  3. Replace the medium on cells with 0.2 ml of growth medium without serum. You may include serum at this step  Add the 54 μl of DNAPlus™-Lipofectamine® complexes (from Step 2d) to each well of cells. Mix gently by rocking the plate back and forth.

  4. Incubate cells at 37°C in a CO2 incubator for 3 hours, then add 0.25 ml of growth medium containing 2X the normal concentration of serum without removing the transfection mixture.

  5. Continue with Step 6 of the Transfection Procedure

Troubleshooting

  1. To prevent cytotoxicity, transfect cultures at a higher confluence, use less Lipofectamine® or DNA, or include serum during transfection.

  2. If cells do not tolerate the absence of serum, transfect in the presence of serum. Prepare complexes for 45 minutes in serum-free medium, then dilute complexes with serum-containing medium before adding to cells. Important: Re-optimize the amount of lipid used.

References

  1. Hawley-Nelson, P., et al. (1993) Focus® 15, 73.

  2. Shih, P., et al. (1997) Focus® 19, 52.
18324.lf.pps      9-Jul-2004