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 plasmidDNA into Hep G2 human hepatocellular carcinoma (ATCC, Cat. No. HB-8065) using Lipofectamine® LTXReagent (Cat No. 15338-100).
Important Guidelines for Transfection
Follow these important guidelines when transfecting DNA into Hep G2 cells using Lipofectamine® LTX Reagent:
- The addition of antibiotics to media during transfection may result in cell death, and has not been tested for Hep G2 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 Hep G2 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:
- Hep G2 cells maintained in RPMI with L-glutamine (Cat. No. 11875-085) supplemented with 10% fetal bovine serum (Cat. No. 26140-079), and 0.1 mM MEM Non-Essential Amino Acids Solution (cat. No. 11140-050). 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
To transfect Hep G2 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 |
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96-well | 0.3 cm2 | 100 μl | 8 x 103 | 20 μl | 100 ng | 0.35 - 0.55 μl | 0.1 μl |
48-well | 1 cm2 | 200 μl | 2 x 104 | 40 μl | 200 ng | 0.7 - 1.1 μl | 0.2 μl |
24-well | 2 cm2 | 500 μl | 4 x 104 | 100 μl | 500 ng | 1.75 - 2.75 μl | 0.5 μl |
12-well | 4 cm2 | 1 ml | 8 x 104 | 200 μl | 1 μg | 3.5 - 5.5 μl | 1.0 μl |
6-well | 10 cm2 | 2 ml | 2 x 105 | 500 μl | 2.5 μg | 8.75 - 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.
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