X-Gal staining of  T98G (Human Brain Glioblastoma) cells 72 hours after transfection by different lots of FuGENE®HD and Competitor Lipofectamine® 2000, 10,000 cells/well

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Reagent / DNA Ratio
0.4μl/0.1μg
8μl Complex per well
Complex Incubation 5 min
Reagent / DNA Ratio
0.5μl/0.2μg
50μl Complex per well
Complex Incubation 20 min

FuGENE®HD

 

Lipofectamine® 2000

 

Reagent / DNA Ratio
0.4μl/0.1μg
8μl Complex per well
Complex Incubation 5 min
Reagent / DNA Ratio
0.5μl/0.2μg
50μl Complex per well
Complex Incubation 20 min

FuGENE®HD

 

Lipofectamine® 2000

 

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

Protocol for T98G (Human Brain Glioblastoma) Cell Transfection by FuGENE HD with gWIZ™ GFP plasmid.

  1. Cell plating

    T98G cells were seeded from 90-100% confluent culture the day before transfection with the density 15,000 cells/well in 100 μl complete growth MEM medium supplemented with 1 mM Sodium Piruvate, 10 mM MEM non-essential Amino Acids Solution and 10% Fetal Bovine Serum.

  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 8 μl of reagent to 160 μl of OptiMEM® / DNA solution.
    3. Mix carefully by pipetting (15 times).
    4. Incubate 5 min at room temperature.
    5. Add 8 μl of complex per well to the cells, and mix thoroughly.
  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.