X-Gal staining of STO cells 24 hours after transfection by FuGENE®HD, FuGENE®6 and Competitor Lipofectamine® 2000

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FuGENE®HD

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

FuGENE®6

Lipofectamine® 2000

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

Protocol for STO Cell Transfection by FuGENE HD

  1. Cell plating

    STO cells were seeded the day before transfection with the density 15,000 cells per well in 100 μl complete growth medium DMEM+10% Fetal 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 plasmid DNA solution in OptiMEM®.
    2. Add 6μl of reagent to 100 μl of OptiMEM® /DNA solution.
    3. Mix carefully by pipetting (10-15 times).
    4. Incubate 5 min at room temperature.
    5. Add 5μl complex per well to the cells, and mix thoroughly.

    Optimal ratio reagent/DNA may vary in range 0.2μl/0.1μg to 0.35μl/0.1μg.

  3. Incubation

    Place the cells into CO2 incubator for 26-28 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.