Lipofectamine® LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. This reference provides a recommended procedure to transfect plasmid DNA into SKBr-3 Human breast carcinoma cells (ATCC Cat. No. HTB-30) using Lipofectamine® LTX Reagent (Cat. No. 15338-100).
Important Guidelines for Transfection
Follow these important guidelines when transfecting DNA into SKBr-3 cells using Lipofectamine® LTX Reagent:
- The addition of antibiotics to media during transfection may result in cell death, and has not been tested for SKBr-3 cells. If you wish to use antibiotics during transfection, test your conditions thoroughly.
- Maintain the same seeding conditions between experiments. Use low-passage cells; make sure that cells are healthy and greater than 90% viable before transfection.
- Transfection can be performed both in the presence or absence of serum. Test serum-free media for compatibility with Lipofectamine® LTX Reagent.
- Using PLUS™ Reagent (Cat. No. 11514-015) enhances transfection performance in SKBr-3 cells.
- We recommend Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) to dilute the DNA and
- Lipofectamine® LTX Reagent before complexing.
- Visit www.lifetechnologies.com/transfection or contact Technical Service for other specialized transfection protocols (including cell-type specific advice on use of PLUS™ Reagent and antibiotics, and a protocol for vector-based RNAi).
- Lipofectamine® LTX Reagent performs well with vector-based RNAi experiments. For siRNA and Stealth™ RNAi transfections, we recommend Lipofectamine® RNAiMAX (Cat. No. 13778-075). Go to www.lifetechnologies.com/RNAi or contact Technical Service for more information.
Have the following reagents on hand before beginning:
- SKBr-3 cells maintained in DMEM supplemented with L-glutamine (Cat. No. 11965-084), 0.1 mM MEM Non-Essential Amino Acids Solution (Cat. No. 11140-050), and 10% Fetal Bovine Serum (Cat. No. 26140-079). Grow cells at 37°C with 5% CO2.
- Plasmid DNA of interest (100 ng/μl or higher)
- Lipofectamine® LTX Reagent (store at +4°C until use), and PLUS™ Reagent (if desired; store at 4°C)
- Opti-MEM® I Reduced Serum Medium
- Appropriate tissue culture plates and supplies
Use this procedure to transfect plasmid DNA into SKBr-3 cells in a 24-well format (for other formats, see Scaling Up or Down Transfections, below). All amounts and volumes are given on a per well basis.
- The day before transfection, trypsinize and count the cells. Plate 4 x 104 cells per well in 0.5 ml of complete growth medium. Cell density should be 50~80% confluent on the day of transfection.
- For each well of cells to be transfected, dilute 0.5 μg of DNA into 100 μl of Opti-MEM® I Reduced Serum Medium without serum.
- If using PLUS™ Reagent: Mix PLUS™ Reagent gently before use, then add 0.5 μl PLUS™ Reagent (a 1:1 ratio to DNA) directly to the diluted DNA. Mix gently and incubate for 5-15 minutes at room temperature.
- For each well of cells, dilute 0.75-1.75 μl of Lipofectamine® LTX into the above diluted DNA solution, mix gently and incubate for 25 minutes at room temperature to form DNA-Lipofectamine® LTX complexes.
- Remove growth medium from cells and replace with 0.5 ml of complete growth medium. Add 100 μl of the DNA-Lipofectamine® LTX complexes directly to each well containing cells and mix gently by rocking the plate back and forth.
- Complexes do not have to be removed following transfection. Incubate the cells at 37°C in a CO2 incubator for 18-24 hours post-transfection before assaying for transgene expression.
To transfect SKBr-3 cells in different tissue culture formats, vary the amounts of Lipofectamine® LTX Reagent, DNA, cells, medium and PLUS™ Reagent used in proportion to the relative surface area, as shown in the table (amounts given on a per well basis).
|Culture vessel|| Surface
|Volume plating medium||Cells per well|| Volume
|96-well||0.3 cm2||100 μl||8 x 103||20 μl||100 ng||0.15 - 0.35 μl||0.1 μl|
|48-well||1 cm2||200 μl||2 x 104||40 μl||200 ng||0.3 - 0.7 μl||0.2 μl|
|24-well||2 cm2||500 μl||4 x 104||100 μl||500 ng||0.75 - 1.75 μl||0.5 μl|
|12-well||4 cm2||1 ml||8 x 104||200 μl||1 μg||1.5 - 3.5 μl||1.0 μl|
|6-well||10 cm2||2 ml||2 x 105||500 μl||2.5 μg||3.75 - 8.75 μl||2.5 μl|
1 Surface areas may vary depending on the manufacturer.
2 If the volume of Lipofectamine® LTX Reagent is too small to dispense accurately, and you cannot pool dilutions, predilute Lipofectamine® LTX Reagent 10-fold in Opti-MEM® I Reduced Serum Medium, and dispense a 10-fold higher amount (should be at least 1.0 μl per well). Discard any unused diluted Lipofectamine® LTX Reagent.