X-Gal staining of PC3 (Prostate Adenocarcinoma) 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
0.25μl/0.1μg
25μl Complex per well
Complex Incubation 20 min

Lipofectamine® 2000

 


Reagent / DNA Ratio
0.25μl/0.1μg
25μl Complex per well
Complex Incubation 20 min
Transfection with competitor reagent was performed according to manufacturer’s instructions

Protocol for PC3 Cell Transfection by FuGENE HD

  1. Cell plating

    PC3 cells were seeded from 90-100% confluent culture with the density 10,000 cells per well in 100μl complete growth medium, DMEM / F12 + 10% 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 0.125μg/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.

    Optimal ratio reagent/DNA may vary in the range 0.25μl/0.1μg to 0.4μl/0.1μg.

  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.