GFP expression in HEK 293 cells transfected with FuGENE®HD, 72 hours incubation, 50,000 cells/well plated.

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Complexes are formed in OptiMEM®

FuGENE®HD

Reagent / DNA Ratio 0.4μl/0.1μg 8μl Complex per well Complex Incubation 5 min

Protocol for 293 Cell Transfection by FuGENE HD

1. Cell plating

   Cells were seeded in 96-well non-tissue culture plate just before transfection with the density 50,000 cells per well in 100 μl “FreeStyle™ 293” growth medium (Invitrogen, Inc.®).

2. Complex preparation (per 20 wells)

   Tissue culture 96-round bottom well plates were used for complex preparation:

   1. Prepare 0.0125μg/μl gWIZ™ GFP DNA solution in OptiMEM® or sterile deionized water.
   2. Add 8 μl of FuGENE®HD to 160μl of DNA solution.
   3. Mix carefully by pipetting (10-15 times).
   4. Incubate 5 min at room temperature.
   5. Add 8 μl of complex per well to the cells, and mix thoroughly.

   Optimal reagent/DNA ratio may vary in the range 0.25μl/0.1μg to 0.45μl/0.1μg.

3. Incubation

   Place the cells into CO₂ incubator for 72 hours.

4. Detection of GFP expression

   Monitor the transgene expression under fluorescent microscope or flowcytometer.