HUVEC (Human umbilical vein endothelial cells) can be succesfully 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 (for example, 5 x 104 cells/well in 48-well plates)
- Optimal confluence for transfection is at 50-60% (visual confluence 90%)
Preparation of lipoplexes
- Mix 0.1 ug plasmid DNA in 15 uL of RPMI
- Mix 0.5 uL of Lipotransfectin in 10 uL of RPMI
- 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 12-16 h at 37º C
- Refresh medium
- Assay tranfection efficiency 24-48 h after transfection