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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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.