X-Gal staining of  A-549 (lung cancer) cells in 96 well plates 24 hours after transfection,  10,000 cells/well.

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FuGENE®HD

Reagent / DNA Ratio

0.3μl/0.1μg

5μl Complex per well

Complex Incubation 10 min

Lipofectamine® LTX

Reagent / DNA Ratio

0.3μl/0.1μg

20μl Complex per well

Complex Incubation 30 min

Lipofectamine® 2000

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 the Transfection of the A-549 (ATCC®: CCL-185™ Human Lung Carcinoma) cells by FuGENE HD in 96 well plates.

  1. Cell Plating

    A-549 cells were seeded from 90-100% confluent culture the day before the transfection with the density 10,000 cells/well in 100μl complete growth medium (F-12K + 10% Fetal Bovine Serum).

  2. Complex preparation (per 20 well)

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

    1. Prepare 0.02μg/μl pCMVβ DNA solution in OptiMEM®.
    2. Add 6 μl of reagent to 100 μl of OptiMEM®/DNA solution.
    3. Mix carefully by pipetting (15 times).
    4. Incubate 10 min at room temperature.
    5. Add 5μl of complex per well to the cells, and mix thoroughly.

    Optimal ratio reagent/DNA may vary in the range 0.25μl/0.1μg to 0.35μl/0.1μg

  3. Incubation

    Transfected cells were incubated in 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.