X-Gal staining of U87 (Glioblastoma-Astrocytoma) cells 72 hours after transfection by FuGENE®HD and Competitor in 96 well plates, 10,000 cell/well

All Complexes are formed in OptiMEM®

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Transfection is done in 100% Fetal Bovine Serum		Transfection is done in growth medium, DMEM +10% Fetal Bovine Serum
Reagent / DNA Ratio 0.25μl/0.1μg 5μl Complex per well Complex Incubation 10 min	FuGENE®HD	Reagent / DNA Ratio 0.25μl/0.1μg 5μl Complex per well Complex Incubation 10 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
Reagent / DNA Ratio 0.3μl/0.1μg 20μl Complex per well Complex Incubation 30 min	Lipofectamine® LTX	Reagent / DNA Ratio 0.3μl/0.1μg 20μl Complex per well Complex Incubation 30 min
Transfection with competitor reagents was performed according to manufacturer’s instructions

Protocol for transfection of the U87 (ATCC®: HTB-14™ Human Brain Glioblastoma-Astrocytoma) Cells by FuGENE HD in 96 well plates

1. Cell plating

   U87 cells were seeded from 90-100% confluent culture the day before transfection with the density 10,000 cells/well in 100 μl complete growth medium (DMEM + 10% Fetal Bovine Serum).

   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 0.02μg/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 10 min at room temperature.
   5. Add 5 μl of complex per well to the cells, and mix thoroughly.

   Optimal ratio of reagent/DNA varies from 0.2μl/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.