Mammalian Cell Transfection - Lipofectamine LTX Reagent
Karmella Haynes, 2012
Updated by Ben Nyer, 2014
- plasmid DNA
- Lipofectamine LTX
- Opti-MEM I Reduced serum medium
- Antibiotic-free growth medium (no pen/strep or other antibiotics)
Procedure - 6-well Format
1 day before transfection:
- Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 at the time of transfection.
- NOTE: if you are using a cell line that does not adhere strongly to the plate (eg, HEK cells), seed the cultures using antibiotic-free medium the day before to avoid having to replace medium the day of transfection.
- At your bench, bring 2.0 μg total plasmid DNA up to 20 μL total volume with fresh, sterile DNase-free water in fresh, sterile 1.5 mL tubes. Take the DNA samples to the tissue culture room.
- In the tissue culture room, warm antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep) at 37°C.
- Warm the Lipofectamine LTX vial, PLUS reagent vial, and and a 6 mL aliquot of Opti-MEM I Reduced Serum Medium to room temperature in the biosafety cabinet.
- Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
- Label sterile microfuge (1.5 ml) tubes.
- Add 570 μL Opti-MEM to each 20 μL DNA sample.
- Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
- Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
- Remove the 6-well plate containing your cultures from the incubator and place it in the biological safety cabinet.
- Aspirate off the old antibiotic-containing medium in the wells. Do NOT scrape the bottom of the wells, as the cells need to remain adherent to the plate.
- Add 4mL of warm antibiotic-free growth medium to each well.
- Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate well of cells.
- Incubate cells at 37°C in a CO2 incubator
- (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.
Transgene expression should be detectable after 18 hours.
For stable cells lines: Passage cells at a 1:10 (or higher, e.g. 1:20, 1:40, etc.) dilution into fresh antibiotic-free growth medium 48 hours after transfection. The next day (after 18 -24 hours later), add selective medium (based on the resistance marker for your plasmid). Grow the cells for 1 - 2 weeks until isolated colonies (about 100 cells per colony) appear. Transfer single colonies into 24-well plates. Grow the cells until they reach 100% confluency. Expand the cells in larger growth vessels (e.g., 6-well plates or 10 cm plates) for further testing.