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X-Gal staining of MCF-7 cells (Invasive Ductal Carcinoma) 48 hours after transfection by FuGENE®HD and Competitor Lipofectamine® 2000, 15,000 cells/well

Complexes are formed in OptiMEM®.

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Transfection is done in
100% Fetal Bovine Serum		 Transfection is done in
Growth medium
DMEM +10%Fetal Bovine Serum

Reagent / DNA Ratio
0.3μl/0.1μg
8μl Complex per well
Complex Incubation 5 min

FuGENE®HD

 
Reagent / DNA Ratio
0.3μl/0.1μg
8μl Complex per well
Complex Incubation 5 min

Reagent / DNA Ratio
0.5μl/0.2μg
50μl Complex per well
Complex Incubation 20 min

Lipofectamine® 2000

 
Reagent / DNA Ratio
0.5μl/0.2μg
50μl Complex per well
Complex Incubation 20 min
 Transfection with competitor reagent was performed according to manufacturer’s instructions

Protocol for MCF-7 Cell Transfection by FuGENE HD

  1. Cell plating

    MCF-7 cells were seeded from 90-100% confluent culture with the density 15000 cells/well in 100μl complete growth medium, DMEM + 10% Fetal Bovine Serum the day before transfection to reach approximately 80% confluence at the time of experiment.

    For transfection in 100% Fetal Bovine Serum the complete growth medium was replaced with Fetal Bovine Serum two hours before transfection.

  2. Complex preparation (per 20 wells)

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

    1. Prepare 0.0125μg/μl pCMVβ DNA solution in OptiMEM®.
    2. Add 6 μl of reagent to 160 μl 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 ratio of Reagent/DNA varies from 0.25μl/0.1μg to 0.35μl/0.1μg.

  3. Incubation.

    Place the cells into CO2 incubator for 48 hours.

  4. Detection of β-gal expression
    1. Remove the medium from the well and wash the cells once with 100μl per well PBS.
    2. Fix the cells in the well with 50μl solution of 4% formaldehyde in PBS for 5min at room temperature.
    3. Wash each well twice with 100μl PBS.
    4. Add 50μl per well of substrate/stain solution and incubate the plate overnight at 37°C.
    5. Observe the cells under microscope and evaluate the proportion of blue (β-gal-positive) cells.