SEAP Expression in COS-7 cells with FuGENE®HD and competitors

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

Protocol for COS-7 Cell Transfection by FuGENE HD

  1. Cell plating
    COS-7 cells were seeded from 90-100% confluent culture the day before transfection with the density 10,000 cells per well in 100 μl 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 0.02 μg/μl gWIZ™ SEAP DNA solution in OptiMEM® or sterile deionized water.
    2. Add 6 μl of reagent to 100 μl of DNA solution.
    3. Mix carefully by pipetting (10-15 times).
    4. Incubate 5 minute at room temperature.
    5. Add 5 μl of complex per well to the cells, and mix thoroughly.

    Optimal ratio of reagent/DNA varies from 0.25μl/0.1μg to 0.4μl/0.1μg.

  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 in 1M Diethanolamine, 1mM MgCl2, pH9.8.
    1. Transfer 5 μl of transfected cells supernatant into a well of flat bottom 96-well plate.
    2. Add 100 μl of PNPP reagent.
    3. Incubate for 5 min at room temperature in the dark place.
    4. Read the plates at 405 nm.