MEF (Mouse Embryonic Fibroblasts) are easily transfected using Lipotransfectin. Here you have the suggested protocol for this specific cell type.
Optimal transfection conditions for your specific application have to be determined experimentally, as they are influenced by the kind of plasmids being transfected and other factors.
- Seed cells around 24 h before tranfection
- Optimal confluence for transfection is at 50-60% (visual confluence 90%)
- Seeding cells 5 h before transfection at a high concentration (0.67 x 105 cells per well in a 12-well plate) has also been reported as a succesfull method
Preparation of lipoplexes
- Mix 1-1.5 ug plasmid DNA in 50 uL of serum-free medium or PBS
- Mix 1-5 uL of Lipotransfectin in 50 uL of serum-free medium or PBS
- Add the DNA solution to the Lipotransfectin solution in this order. It is very important to do it in this order.
- Mix gently by pipetting up and down once
- Incubate solution at room temperature for 15 – 20 min.
- Add as soon as possible the lipoplex solution dropwise to the cells and swirl the flask with extreme care.
- Incubate cells for 24-48 h at 37º C. Refreshing medium 4-6 h after adding the lipoplex solution can be beneficial in some cases.