Invitrogen™ 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 HEK 293, human embryonic kidney cells (ATCC No. CRL-1573) using Lipofectamine LTX Reagent.
Important guidelines for transfection
Follow these important guidelines when transfecting HEK 293 cells using Lipofectamine LTX Reagent:
- Maintain the same seeding conditions between experiments. Use low-passage cells; make sure 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.
- We recommend Gibco™ Opti-MEM™ I Reduced Serum Medium (Cat. No. 31985-070) to dilute the DNA Lipofectamine LTX Reagent before complexing.
- Visit www.lifetechnologies.com/genedelivery or contact Technical Services for other specialized transfection protocols.
- Lipofectamine LTX Reagent performs well with vector-based RNAi experiments. For Invitrogen™ siRNA and Stealth RNAi™ transfections, we recommend Invitrogen™ Lipofectamine™ RNAiMAX Transfection Reagent. Go to www.lifetechnologies.com/RNAi or contact Technical Service for more information.
Have the following reagents on hand before beginning:
- HEK 293 cells maintained in Gibco™ Dulbecco's Modified Eagle Medium (DMEM) (Cat. No. 11960-044) supplemented with 4 mM Gibco™ L-Glutamine (Cat. No. 25030-081), 10% Gibco™ Fetal Bovine Serum (Cat No. 16000-044). Grow cells at 37o C with 5% CO2.
- Plasmid DNA of interest.
- Lipofectamine LTX Reagent (store at +4oC until ready to use)
- Opti-MEM I Reduced Serum Media
- Appropriate tissue culture plates and supplies
- The day before transfection, trypsinize and count the cells. Plate 0.5 -1.25x105 cells per well in 0.5 mL of complete growth medium. Cell density should be 50–80% confluent on the day of transfection.
- (Optional) The day of transfection, remove growth medium from cells and replace with 0.5 mL of complete growth medium.
- For each well of cells to be transfected, dilute 0.5 μg of DNA in 100 μL of Opti-MEM I Reduced Serum Media without serum.
- For each well of cells, add 0.75-1.75 μL of Lipofectamine LTX Reagent into the above diluted Opti-MEM:DNA solution, mix gently and incubate 30 minutes at room temperature to form DNA- Lipofectamine LTX Reagent complexes.
- After 30 minute incubation, add 100 μl of the DNA- Lipofectamine LTX Reagent 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 37oC in a CO2 incubator for 18–24 hours post-transfection before assaying for transgene expression.
|Volume plating medium||Cells per well||Volume
|96-well||0.3 cm2||100 μl||2.5 x 104||20 μl||100 ng||0.15 - 0.35 μl|
|48-well||1 cm2||200 μl||5 x 104||40 μl||200 ng||0.30 - 0.7 μl|
|24-well||2 cm2||500 μl||1.25 x 105||100 μl||500 ng||0.75 - 1.75 μl|
|12-well||4 cm2||1 ml||2.5 x 105||200 μl||1 μg||1.5 - 3.5 μl|
|6-well||10 cm2||2 ml||6.25 x 105||500 μl||2.5 μg||3.75 - 8.75 μl|