1) Start With Healthy Cells
a. Passage cells 3–4 times after thawing before using them in transfection experiments. This gives the cells time to recover from the thawing procedure and return to their normal rate of growth.
b. Only use cells >90% viability—you can easily determine cell viability using Trypan Blue Stain.
c. Passage cells on a regular basis and do not allow the cells to become confluent or overgrown. Allowing cells to become confluent may change their growth rate as well as cell morphology.
i. Passage cells at or before 90% confluency. We recommend using TrypLE™ reagent for detachment of cells. TrypLE™ reagent can be stored in the hood at room temperature, and you can skip the PBS wash by adding extra TrypLE™ reagent and aspirating all but enough solution to cover the surface of the flask.
ii. Passage conditions are dependent on the individual cell line. Some rules of thumb:
- For fast-growing cells, with a doubling time of every 16 hrs (e.g., HEK-293), split cells 1:10
- For slow-growing cells, with a doubling time of every 36 hrs (e.g., primary cells), split cells 1:5
d. Maintain frozen stocks of cell lines and regularly thaw new cells. Cells can change growth rate and morphology at high passage numbers (>30–40).
2) Plan Your Experiment Before Going Into the Lab.
It is important to familiarize yourself with the protocol, determine how much material you need, and confirm that you have everything you need before getting started.
a. Start by designing a plate map outlining each treatment or experimental condition.
b. Calculate the amount of lipid and DNA stock you will need, and confirm that you have enough of the following materials: Opti-MEM® medium, Lipofectamine® 2000 or Lipofectamine® LTX reagent, and DNA (0.5–1 µg/µL).
3) Use High-quality DNA in Your Transfection.
a. Prepare DNA using an endotoxin-free kit or protocol.
b. Determine the DNA purity by measuring the OD 260/280 ratio, which should be between 1.7–1.9. Higher or lower ratios indicate impurities and should not be used in transfection experiments.
c. Dilute DNA in DNase/RNase-free water or TE.
d. Prepare working concentrations of 0.5–1 µg/µL. DNA can be concentrated by SpeedVac® concentrator or diafiltration (e.g., Amicon® Ultra filtration).
4) Plate Your Cells the Day Before You Plan to Transfect.
a. If plated >1 day prior to transfection, the transfection efficiency may decrease.
b. Seed your cells at a density such that they will be 70–90% confluent at the time of transfection.
c. Cell density at the time of transfection affects the transfection efficiency. To simplify transfection optimization, or to save time, we recommend plating at 2 different densities to ensure a high transfection efficiency.
d. Cell plating and transfection can be performed at the same time or by using a reverse transfection protocol. In a reverse transfection, you need to use 2.5 times more cells than you would use in a regular or forward transfection.
e. Transfection complexes can be added to cells in media containing antibiotics and serum without impacting the transfection efficiency.
5) Prepare Lipid DNA Complexes.
a. For maximum performance, we recommend complexing the lipid and DNA in Opti-MEM® medium. Alternatively, a serum-free medium can be used.
b. Dilute the lipid in Opti-MEM® medium. Dilute the DNA in a second tube of Opti-MEM® medium, and mix equal volumes to complex the lipid and DNA. The 2-step dilution method results in higher quality data and generates more reproducible results compared to adding the lipid directly to the diluted DNA.
c. Once the lipid is diluted in Opti-MEM® medium, the optimal incubation time is 2 to 5 min before adding the diluted DNA. Do not incubate the diluted lipid for longer than 20 min.
d. The DNA Opti-MEM® solution is more stable and can be prepared up to 4 hrs in advance but not longer.
e. Once equal volumes of the diluted lipid and DNA have been combined, mix the solution by pipetting up and down slowly, flicking the bottom of the tube, or vortexing quickly.
f. After the lipid DNA complex has incubated for 5 to 10 min, transfer the complex to the well containing cells and growth medium. Add complexes drop-wise on top of the medium and gently rock the plate after adding to the mix. Avoid forceful dispensing of lipid DNA complex in the well because it may displace cells.
6) Use a positive control such as GFP or LacZ reporter plasmid to assess transfection efficiency.
The number or percent of cells transfected and the intensity of GFP expression can easily be determined using microscopy (e.g., FLoid™ cell imaging) or flow cytometry (e.g., an Attune® cytometer). LacZ expression can be determined using ToxBLAzer™ kits.
a. Plasmid expression should be visible or detectable 24 to 48 hrs post-transfection.
b. The positive control can be in a separate well or co-transfected with your plasmid of interest. For co-transfection, you can mix 50–100 ng of GFP plasmid with 100 ng of your plasmid of interest together in Opti-MEM® medium prior to adding the diluted lipid.
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