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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 HT-1080 Human Fibrosarcoma Cells (ATCC Cat. No. CCL-121) using Lipofectamine® LTX Reagent (Cat. No. 15338-100).
Important Guidelines for Transfection
Follow these important guidelines when transfecting DNA into HT-1080 cells using Lipofectamine® LTX
Reagent:
- The addition of antibiotics to media during transfection may result in cell death, and has not been tested for HT-1080 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 cell 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 HT-1080 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.
Materials Needed
Have the following reagents on hand before beginning:
- HT-1080 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
.
TOP
Use this procedure to transfect plasmid DNA into HT-1080 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.
TOP- The day before transfection, trypsinize and count the cells. Plate 8 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 0.75-2.75 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 HT-1080 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 area per well1 | Volume plating medium | Cells per well | Volume dilution medium2 | DNA | Lipofectamine® LTX Reagent | PLUS™ Reagent |
|---|---|---|---|---|---|---|---|
| 96-well | 0.3 cm2 | 100 μl | 1.6 x 104 | 20 μl | 100 ng | 0.15 - .75 μl | 0.1 μl |
| 48-well | 1 cm2 | 200 μl | 4 x 104 | 40 μl | 200 ng | 0.3 - 1.5 μl | 0.2 μl |
| 24-well | 2 cm2 | 500 μl | 8 x 104 | 100 μl | 500 ng | 0.75 - 2.75 μl | 0.5 μl |
| 12-well | 4 cm2 | 1 ml | 1.6 x 105 | 200 μl | 1 μg | 1.5 - 5.5 μl | 1.0 μl |
| 6-well | 10 cm2 | 2 ml | 4 x 105 | 500 μl | 2.5 μg | 3.75 - 13.75 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.
25-0937W 9 Jun 2006