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

Lipofectamine® 2000

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

Lipofectamine® 2000

Reagent / DNA Ratio
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.