X-Gal staining of MCF-7 (Invasive Ductal Carcinoma) cells 72 hours after transfection by different reagents at low concentration of complexes

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FuGENE®HD

Reagent / DNA Ratio 0.0625μl/0.025μg 5μl Complex per well Complex Incubation 5 min

Lipofectamine® 2000

Reagent / DNA Ratio 0.125μl/0.05μg 12.5μl Complex per well Complex Incubation 20 min

Lipofectamine® 2000

Reagent / DNA Ratio 0.25μl/0.1μg 25μl Complex per well Complex Incubation 20 min

Transfection with competitive reagent was performed according to manufacturer’s instructions

Protocol for Transfection of MCF-7 Cells by FuGENE HD with pCMVβ in 96-well plate (Low complex concentration).

1. Cell plating.

   MCF-7 cells were seeded from 90-100% confluent culture with the density 15000 cells/well in 100μl complete growth medium, DMEM + 10% Fetal Bovine Serum the day before transfection to reach approximately 80% confluence at the time of experiment.

2. Complex preparation (per 40 wells)
   Tissue culture 96-round bottom well plates were used for complex preparation:
   1. Prepare 0.005μg/μl pCMVβ DNA solution in OptiMEM®.
   2. Add 2.5 μl of FuGENE® HD to 200 μl of DNA solution.
   3. Mix carefully by pipetting (10-15 times).
   4. Incubate complex for 5 min at room temperature.
   5. Add 5 μl of complex per well to the cells, and mix thoroughly.
3. Incubation.

   Place the cells into CO₂ incubator for 72 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.