X-Gal staining  of STSAR90 (Osteo-Sarcoma) cells 72 hours after transfection by FuGENE®HD, FuGENE®6 and Competitor Lipofectamine® 2000, 10,000 cells/well.

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100% Fetal Bovine Serum		Growth medium DMEM/F12 +15% Fetal Bovine Serum
Reagent / DNA Ratio 0.3μl/0.1μg 8μl Complex per well Complex Incubation 5 min	FuGENE®HD	Reagent / DNA Ratio 0.3μl/0.1μg 8μl Complex per well Complex Incubation 5 min
Reagent / DNA Ratio 0.3μl/0.1μg 8μl Complex per well Complex Incubation 5 min	FuGENE®HD	Reagent / DNA Ratio 0.3μl/0.1μg 8μl Complex per well Complex Incubation 5 min
Reagent / DNA Ratio 0.3μl/0.1μg 10μl Complex per well Complex Incubation 15 min	FuGENE®6	Reagent / DNA Ratio 0.3μl/0.1μg 10μl Complex per well Complex Incubation 15 min
Reagent / DNA Ratio 0.5μl/0.2μg 50μl Complex per well Complex Incubation 20 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 STSAR90 (Osteo Sarcoma) Cell Transfection by FuGENE HD

1. Cell plating

   STSAR90 cells were seeded from 90-100% confluent culture with the density 10,000 cells/well in 100μl complete growth medium, DMEM / F12+ 15% Fetal Bovine Serum the day before transfection to reach approximately 80% confluence at the time of experiment.

   For transfection in 100% Fetal Bovine Serum the complete growth medium was replaced with Fetal Bovine Serum two hours before transfection.

2. Complex preparation (per 20 wells)

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

   1. Prepare 0.0125μg/μl pCMVβ DNA solution in OptiMEM®.
   2. Add 6 μl of FuGENE®HD to 160 μl of DNA solution.
   3. Mix carefully by pipetting (10-15 times).
   4. Incubate complex for 5 min at room temperature.
   5. Add 8 μl of complex per well to the cells, and mix thoroughly.

   Optimal ratio reagent/DNA may vary in the range 0.25μl/0.1μg to 0.4μl/0.1μg.

3. Incubation

   Transfected cells were incubated in 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.