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 Primary Mouse Neural Progenitor Cells (mNPC) using Lipofectamine LTX Reagent (Cat. No. 15338-100).
Important Guidelines for Transfection
Follow these important guidelines when transfecting mNPC 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.
- NCP’s grow as neurospheres, and can be adhered to culture plates and differentiated into neurons,
astrocytes, and oligodendrocytes upon addition of serum and insulin-like growth factor.
- Transfection can be performed both in the presence or absence of serum. Test serum-free media for compatibility with Lipofectamine LTX Reagent.
- Using PLUS Reagent (Cat. No. 11514-015) enhances transfection performance in Primary Mouse Neural Progenitor Cells.
- 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.
- 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.
Have the following reagents on hand before beginning:
- Primary Mouse Neural Progenitor (NCP) cells maintained in DMEM (GIBCO Cat. No. 11960-044) medium
supplemented with 1% L-glutamine (GIBCO Cat. No. 25030), 1% penicillin-streptomycin (Cat. no. 15070-063), 1% dextrose, and epidermal growth factor. 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), and PLUS™ Reagent (store at 4°C)
- Opti-MEM I Reduced Serum Medium
- Appropriate tissue culture plates and supplies
Use this procedure to transfect plasmid DNA into Primary Mouse Neural Progenitor Cells in a 24-well format. All amounts and volumes are given on a per well basis.
- Begin transfection one or seven days after differentiation in 24-well plate.
- The day of transfection, remove growth medium from cells and replace with 0.5 ml of complete growth medium without antibiotics.
- For each well of cells to be transfected, dilute 0.25, 0.50, or 1.0 μg of DNA into 100 μl of Opti-MEM I Reduced Serum Medium without serum.
- Mix PLUS™ Reagent gently before use, then add 2.5 μl PLUS™ Reagent directly to the diluted DNA. Mix gently and incubate for 5-15 minutes at room temperature.
- For each well of cells, add 0.1-2.50 μl of Lipofectamine LTX Reagent into the above diluted DNA solution, mix gently and incubate for 30 minutes at room temperature to form DNA-Lipofectamine LTX complexes.
- After the 30 minute incubation, 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.
- 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.
- Results are best for NPC’s differentiated for 1 day (compared to 7-day differentiated).
- Combinations that gave best results:
1.5 μl Lipofectamine LTX / 2.5 μl PLUS/ 1.0 μg plasmid
2.5 μl Lipofectamine LTX/ 2.5 μl PLUS/ 500 ng plasmid