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-29, human colon adenocarcinoma cells (ATCC No. HTB-38) using Lipofectamine LTX® Reagent.
Important Guidelines for Transfection
Follow these important guidelines when transfecting HT-29 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 Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-070) to dilute the DNA Lipofectamine LTX® Reagent before complexing.
- Using PLUS™ Reagent (Cat. No. 11514-015) enhances transfection performance in HT-29 cells
- 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 siRNA and Stealth RNAi transfections, we recommend Lipofectamine RNAiMAX. Go to www.lifetechnologies.com/RNAi or contact Technical Service for more information.
Have the following reagents on hand before beginning:
- HT-29 cells maintained in RPMI Medium 1640 (Cat. No. 21870-076) supplemented with 4 mM L-Glutamine (Cat. No. 25030-081), 10% fetal bovine serum (Cat. No. 16000-044 ). Grow cells at 37o C with 5% CO2
- Plasmid DNA of interest
- Lipofectamine LTX® Reagent (store at +4o C until ready to use) , and PLUS™ Reagent (if desired; store at 4° C)
- Opti-MEM® I Reduced Serum Media
- Appropriate tissue culture plates and supplies
- The day before transfection, trypsinize and count the cells. Plate 1.5 x 105 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 0.75 μg of DNA in 100 μl of Opti-MEM® I Reduced Serum Media without serum.
- If using PLUS™ Reagent: Mix PLUS™ Reagent gently before use, then add 0.75 μl PLUS™ Reagent (a 1:1 ratio to DNA) directly to the diluted DNA. Mix gently and incubate 5-15 minutes at room temperature.
- For each well of cells, add 3.75-5.0 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.
|Culture vessel|| Surface
|Volume plating medium||Cells per well|| Volume
|96-well||0.3 cm2||100 μl||3.0 x 104||20 μl||150 ng||0.75 - 1.0 μl||0.15 μl|
|48-well||1 cm2||200 μl||6.0 x 104||40 μl||300 ng||1.5 - 2.0 μl||0.3 μl|
|24-well||2 cm2||500 μl||1.5 x 105||100 μl||750 ng||3.75 - 5 μl||0.75 μl|
|12-well||4 cm2||1 ml||3.0 x 105||200 μl||1.5 μg||7.5 - 10 μl||1.5 μl|
|6-well||10 cm2||2 ml||7.5 x 105||500 μl||3.75 μg||18.75 - 25 μl||3.75 μl|