Procedure for Transfection of Mammalian Cells

Materials:

	Lipofectamine
	IMDM containing 10% fetal bovine serum, 1% glutamine, 1% aa
	IMDM containing 1% glutamine
	IMDM containing 20% fetal bovine serum, 1% glutamine, 1% aa

1. In a six-well or 35 mm tissue culture plate, seed ~2x 10⁵ cells per well in 2 ml IMDM containing 10% FBS and nonessential amino acids.
2. Incubate the cells at 37E C in a CO₂ incubator until the cells are 70-80% confluent. This will usually take 18-24 h.
3. Prepare the following solutions in 12 x 75 mm sterile tubes:
   Solution A: For each transfection, dilute 2 μg DNA (plasmid) in 375 μl serum-free IMDM (containing nonessential amino acids).
   Solution B: For each transfection, dilute 12 μl LIPOFECTAMINE Reagent in 375 μl serum-free IMDM.
4. Combine the two solutions, mix gently, and incubate at room temperature for 15-45 min. The solution may appear cloudy, however this will not impede the transfection.
5. Wash the cells once with 2 ml serum-free IMDM.
6. For each transfection, add 750 μl serum-free IMDM to each tube containing the lipid-DNA complexes. Do not add antibacterial agents to media during transfection. Mix gently and overlay the diluted complex solution onto the washed cells.
7. Incubate the cells for 5 h at 37E C in a CO₂ incubator.
8. Add 1.5 ml IMDM with 20% FBS without removing the transfection mixture. If toxicity is a problem, remove the transfection mixture and replace with normal growth medium.
9. Replace medium at 18-24 h following start of transfection.
10. Assay cell extracts for gene activity 24-72 h after the start of transfection, depending on cell type and promoter activity.