Transfecting Plasmid DNA into GripTite™ 293 MSR Cells Using Lipofectamine® LTX Reagent

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Introduction

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 GripTite™ 293 MSR Human Embryonic Kidney Cells (Invitrogen Cat. No. R795-07) using Lipofectamine® LTX Reagent (Cat. No. 15338-100).

Important Guidelines for Transfection

Follow these important guidelines when transfecting DNA into GripTite™ 293 MSR cells using Lipofectamine®
LTX Reagent:

  • The addition of 600 μg/ml Geneticin® (Catalog No. 10131-027) and 100 u/ml Penicillin/ 100 μg/ml Streptomycin (Catalog No. 15140-122) to media during transfection does not result in cell death for GripTite™ 293 MSR 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/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:

  • GripTite™ 293 MSR 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), 10% Fetal Bovine Serum (Cat. No. 26140- 079), and 600 μg/ml Geneticin® (Catalog No. 10131-027). 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 GripTite™ 293 MSR Cells

Use this procedure to transfect plasmid DNA into GripTite™ 293 MSR 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 2 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.
  2. 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.
  3. For each well of cells, dilute 1.5-3.5 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 GripTite™ 293 MSR 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
96-well0.3 cm2100 μl4 x 10420 μl100 ng0.3 - 0.7 μl
48-well1 cm2200 μl1 x 10540 μl200 ng0.6 - 1.4 μl
24-well2 cm2500 μl2 x 105100 μl500 ng1.5 - 3.5  μl
12-well4 cm21 ml4 x 105200 μl1 μg3.0 - 7.0 μl
6-well10 cm2 2 ml1 x 106500 μl2.5 μg7.5 - 17.5 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. 

25-0932W       9 Jun 2006