Transfecting Plasmid DNA into NIH3T3 Cells Using Lipofectamine™ LTX Reagent

实验概要

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  NIH3T3 Mouse Embryonic Fibroblasts (ATCC Cat. No. CRL-1658) using  Lipofectamine™ LTX Reagent (Cat. No. 15338-100).

主要试剂

NIH3T3 cells maintained in DMEM supplemented with L-glutamine [Details: Life Technologies, Cat. No. 11965-084]

0.1 mM MEM Non-Essential Amino Acids Solution [Details: Life Technologies, Cat. No. 11140-050]

10% Fetal Bovine Serum [Details: Life Technologies, Cat. No. 26140-079]

Plasmid DNA of interest (100 ng/μl or higher)

Lipofectamine™ LTX Reagent

PLUS™ Reagent [Details: Life Technologies, Cat. No. 13778-075]

Opti-MEM® I Reduced Serum Medium[Details: Life Technologies, Cat. No. 31985-062]

Appropriate tissue culture plates and supplies.

实验步骤

Use  this procedure to transfect plasmid DNA into NIH3T3 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 0.5 μg of DNA into 100  μl of Opti-MEM® I Reduced Serum Medium without serum.

3.         If using PLUS™ Reagent: Mix PLUS™ Reagent gently before use, then add  0.5 μl PLUS™ Reagent (a 1:1 ratio to DNA) directly to the diluted DNA.  Mix gently and incubate for 5-15 minutes at room temperature.

4.         For each well of cells, dilute 0.75-2.25 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.

5.         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.

6.        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.

注意事项

Follow these important guidelines when transfecting DNA into NIH3T3 cells using Lipofectamine™ LTX Reagent:

1.         The addition of antibiotics to media during transfection may result in  cell death, and has not been tested for NIH3T3 cells. If you wish to use  antibiotics during transfection, test your conditions thoroughly.

2.         Maintain the same seeding conditions between experiments. Use  low-passage cells; make sure that cells are healthy and greater than 90%  viable before transfection.

3.         Transfection can be performed both in the presence or absence of serum.  Test serum-free media for compatibility with Lipofectamine™ LTX  Reagent.

4.        Using PLUS™ Reagent (Cat. No. 11514-015) enhances transfection performance in NIH3T3 cells.

5.         We recommend Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) to  dilute the DNA and Lipofectamine™ LTX Reagent before complexing.

6.         Visit www.invitrogen.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).

7.         Lipofectamine™ LTX Reagent performs well with vector-based RNAi  experiments. For siRNA and Stealth™ RNAi transfections, we recommend  Lipofectamine™ RNAiMAX (Cat.No.13778-075).