X-Gal staining of A-375 (Melanoma, Skin Cancer) cells 72 hours after transfection by FuGENE®HD and Competitor Lipofectamine® 2000, 15,000 cells/well.

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FuGENE®HD

Lipofectamine® 2000


Reagent / DNA Ratio
0.3μl/0.1μg
8μl Complex per well
Complex Incubation 10 min
Complexes are formed in Sterile Deionized Water
Reagent / DNA Ratio
0.5μl/0.2μg
50μl Complex per well
Complex Incubation 20 min

Reagent / DNA Ratio
0.3μl/0.1μg
8μl Complex per well
Complex Incubation 10 min
Complexes are formed in
OptiMEM®

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

Reagent / DNA Ratio
0.3μl/0.1μg
8μl Complex per well
Complex Incubation 10 min
Complexes are formed in DMEM with 1.5g/L sodium bicarbonate
+ 10% Fetal Bovine Serum

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

FuGENE®HD

 

Lipofectamine® 2000

 

Transfection with competitor reagent was performed according to manufacturer’s instructions

Protocol for A-375 Cell Transfection by FuGENE HD with pCMVβ plasmid.

  1. Cell plating

    A-375 cells were seeded from 90-100% confluent culture the day before transfection with the density 15,000 cells per well in 100 μl complete growth medium DMEM with 1.5 g/L sodium bicarbonate +10% Fetal Bovine Serum in 96-well plates. A-375 cells were at approximately 80% confluence on a day of 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®, sterile deionized water or growth medium.
    2. Add 6 μl of reagent to 100 μl of DNA solution.
    3. Mix carefully by pipetting (15 times).
    4. Incubate 10 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.