X-Gal staining of RAW (Abelson murine leukemia virus-induced tumor cells) cells 24 hours after transfection in 96 well plates.

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RAW 50,000/well

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 RAW cells by FuGENE®HD in 96 well plates.

  1. Cell Plating

    RAW cells were seeded from 90-100% confluent culture the day before the transfection with the density 50,000 cells/well in 100μl complete growth medium DMEM + 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 5 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.