X-Gal staining of primary rat hepatocytes 48 hours after transfection by FuGENE®HD and FuGENE®6. Transfection was performed 4 days after seeding and shipping.

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Reagent / DNA Ratio
5μl Complex per well
Complex Incubation 15 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 Primary Rat Hepatocytes Cell Transfection by FuGENE HD

  1. Cell plating

    Primary Rat Hepatocytes were obtained from LifeTechnologies® GIBCO™ cat# RTFN96. Cells were cultivated in 96well plates on Collagen type I matrix without overlay. The transfection experiments were conducted 4 days after cell seeding.

  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 4 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 5μl 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 48 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.