HeLa S3 Cells


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 DNA into HeLa S3 cells using Lipofectamine® LTX Reagent:

  • The addition of 100 U/ml Penicillin/100 μg/ml Streptomycin (Catalog No. 15140-122) to media during transfection does not result in cell death for HeLa S3 cells. If you wish to use other higher concentrations or other 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.
  • 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:

  • HeLa S3 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)
  • Opti-MEM® I Reduced Serum Medium
  • Appropriate tissue culture plates and supplies

Transfection of HeLa S3 Cells

Use this procedure to transfect plasmid DNA into HeLa S3 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.

  1. 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.
  2. For each well of cells to be transfected, dilute 0.25 μg of DNA into 100 μl of Opti-MEM® I Reduced Serum Medium without serum.
  3. For each well of cells, dilute 0.5-1.25 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.
  4. 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.
  5. 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.

Scaling Up or Down Transfections

To transfect HeLa S3 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
Volume plating medium Cells per well Volume
DNA Lipofectamine®
LTX Reagent
96-well 0.3 cm2 100 μl 8 x 103 20 μl 50 ng 0.1 - 0.25 μl
48-well 1 cm2 200 μl 2 x 104 40 μl 100 ng 0.2 - 0.5 μl
24-well 2 cm2 500 μl 4 x 104 100 μl 250 ng 0.5 - 1.25  μl
12-well 4 cm2 1 ml 8 x 104 200 μl 500 ng 1.0 - 2.5 μl
6-well 10 cm2 2 ml 2 x 105 500 μl 1.25 μg 2.5 - 6.25 μl

25-0934W   9  Jun  2006