X-Gal staining of HighFive™ cells 24 hours after transfection by FuGENE®HD and FuGENE®6

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Reagent / DNA Ratio
5μl Complex per well
Complex Incubation 0 min


Reagent / DNA Ratio
10μl Complex per well
Complex Incubation 15 min

Protocol for HighFive™ Cell Transfection by FuGENE®HD

  1. Cell plating

    HighFive™ cells were seeded from 90-100% confluent culture the day before transfection  with the density 20,000 cells per well in 100 μl complete growth medium (ExpressFive®, Life Technologies™) in 96-well plates. HighFive™ cells were at approximately 80% confluence on the day of transfection.

  2. Complex preparation (per 20 wells)

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

    1. Prepare β-gal expressing insect reporter plasmid DNA solution (pIBHis-LacZ) in deionized water, 0.01 μg/μl.
    2. Add 3.5 μl of reagent to 100 μl of DNA solution.
    3. Mix carefully by pipetting (5-10 times).
    4. Incubation of complex is not necessary.
    5. Add 5μl complex to the cells, per well and mix thoroughly.

    Optimal ratio FuGENE HD/DNA varies from 0.15µl/0.05µg to 0.25µl/0.05µg.

  3. Incubation

    Place the cells into 27oC 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.