X-Gal and SEAP expression in STO cells 24 hours after transfection with different agents

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FuGENE®HD


Reagent / DNA Ratio
0.3μl/0.1μg
5μl Complex per well
Complex Incubation 5 min

Lipofectamine® 2000


Reagent / DNA Ratio
0.5μl/0.2μg
50μl Complex per well
Complex Incubation 20 min

FuGENE®6


Reagent / DNA Ratio
.03μl/0.1μg
10μl Complex per well
Complex Incubation 15 min
Seap-Expression-Chart
Transfection with competitive reagent was performed according to manufacturer’s instructions

Protocol for STO Cell Transfection by FuGENE HD

  1. Cell plating.

    STO cells were seeded the day before transfection with the density 15,000 cells per well in 100μl complete growth medium DMEM+10% Fetal Bovine Serum (Gibco®).

  2. Complex preparation (per 20 wells)
    Tissue culture 96-round bottom well plates were used for complex preparation:
    1. Prepare gWIZ™ SEAP DNA solution in OptiMEM® or sterile deionized water, 0.0125μg/μl.
    2. Add 5 μl of reagent to 160 μl of DNA solution.
    3. Mix carefully by pipetting (15 times).
    4. Incubate complex for 5-10 min at room temperature.
    5. Add 8 μl of complex per well to the cells, and mix thoroughly.
  3. Incubation.

    Place the cells into CO2 incubator for 24 hours.

  4. Detection of SEAP expression

    SEAP activity is quantitatively determined by a colorimetric method based on hydrolysis of chromogenic substrate para-nitrophenyl phosphate (PNPP). 1 μg/μl of PNPP is prepared in1M Diethanolamine, 1mM MgCl2 pH9.8

    1. Pipette 11μl supernatant of transfected cells into a well of flat bottom 96-well plate.
    2. Add 100μl of PNPP solution.
    3. Wash each well twice with 100μl PBS.
    4. Incubate for 10 min at room temperature in the dark place.
    5. Read the plates at 405 nm