X-Gal staining of NIH3T3 cells 24 hours after transfection by FuGENE®HD, FuGENE®6 and competitor Lipofectamine® 2000, 5,000 cells/well in 96 well plate

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Reagent / DNA Ratio
5μl Complex per well
Complex Incubation 15 min

Lipofectamine® 2000


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



Reagent / DNA Ratio
10μl Complex per well
Complex Incubation 15 min
 Transfection with competitor reagent was performed according to manufacturer’s instructions

Protocol for NIH/3T3 Cell Transfection by FuGENE HD

  1. Cell plating

    NIH3T3 cells were seeded the day before transfection with the density 5,000 cells per well in 100 μl complete growth medium DMEM+10% Calf Bovine Serum.

  2. Complex preparation (per 20 wells)

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

    1. Prepare 0.02μg/μl pCMVβ DNA solution in OptiMEM®.
    2. Add 5 μl of reagent to 100 μl of OptiMEM® /DNA solution.
    3. Mix carefully by pipetting (10-15 times).
    4. Incubate 15 min at room temperature.
    5. Add 5ml complex per well to the cells, and mix thoroughly.

    Optimal ratio reagent/DNA may vary in range 0.2μl/0.1μg to 0.3μl/0.1μg

  3. Incubation

    Place the cells into CO2 incubator for 24 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.