Lipofectin Transfection of 1° Neonatal Cardiocytes
1) Lipofectin reagent - 1 mg/ml BRL cat. no. 82925A store at 4°C
2) Bi°Coat Tissue Culture Plates 60mm Dish - Collab. Research cat. no. 40401
3) Opti-MEM-Gibco cat. no. 320-1985AJ store at 4°C in dark
4) 12 X 75 mm polystyrene sterile tabes - Falcon cat. no. 2003
1. Plate 2 X 106 cells on 60 mm collagen-coated tissue culture plate (Collab Res).
2. 24 hr. after plating change media to new nutrient media (see myocyte isolation protocal)
3. The following AM (36 h post isolation). Spin down DNA1, sterilly wash with ETOH and dry in laminar flow hood.
4. Add 50 ug (50 ul) of lipofectin reagent2 to 1.5 ml Opti-MEM in polystyrene snap cap sterile tube. Resuspend DNA in 100 ul of Opti-MEM and add to 1.4 ml of Opti- MEM in a separate polystyrene snap-cap tube. Add DNA to lipofectin and mix by inversion.
5. Pipette off nutrient media from cells and add 3 ml of Opti-MEM/DNA/lipofectin to cells.
6. Incubate 37°C, 5% CO2 X 5 hr3
7. Remove Opti-MEM. Add back nutrient media.
8. Harvest at 60 h
1. The amount of DNA (2-20 ug) must be optimized to the cell type.
2. The amount of lipofectin must be optimized to the cell type.
3. The optimal time of exposure to lipofectin reagent must be empirically determined.