X-Gal staining  of SCC61 (Head and Neck Cancer) cells 72 hours after transfection by FuGENE®HD, FuGENE®6 and Competitor Lipofectamine® 2000, 10,000 cells/well.

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100% Fetal Bovine Serum

Growth medium DMEM/F12+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
0.3μl/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
0.3μl/0.1μg
10μl Complex per well
Complex Incubation 15 min

FuGENE®6

 


Reagent / DNA Ratio
0.3μl/0.1μg
10μl Complex per well
Complex Incubation 15 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 SCC61 (Head & Neck) Cell Transfection by FuGENE HD

  1. Cell plating

    SCC61 cells were seeded from 90-100% confluent culture with the density 10,000 cells per well in 100μl complete growth medium, DMEM / F12 + 20% Fetal Bovine Serum the day before experiment to reach approximately 80% confluence at the time of transfection.

    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 OptiMEM® / DNA solution.
    3. Mix carefully by pipetting (15 times).
    4. Incubate 5 min at room temperature.
    5. Add 8 μl of complex per well to the cells, and mix thoroughly.
  3. Incubation

    Transfected cells were incubated in CO2 incubator for 72 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.