SEAP Expression in CHO-K1 cells with FuGENE®HD and competitors

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

Protocol for CHO-K1 Cell Transfection by FuGENE HD

  1. Cell plating
    CHO-K1 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 F-12K with 10% fetal bovine serum Gibco®.
  2. Complex preparation (per 20 wells)
    Tissue culture 96-well round bottom plates were used for complex preparation:
    1. Prepare gWIZ SEAP DNA solution in Opti-MEM®I or sterile deionized water, 0.0125μg/μl.
    2. Add 160 μl of DNA solution per well.
    3. Mix carefully by pipetting (10-15 times).
    4. Incubate 5-10 minute at room temperature.
    5. Add 8 μl of complex per well to the cells, and mix thoroughly.Optimal ratio of reagent/DNA varies from 0.2μl/0.1μg to 0.35μl/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 mg/ml of PNPP is prepared in1M Diethanolamine, 1mM MgCl2, pH9.8.
    1. Pipette 10 μl supernatant of transfected cells 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.