- 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®.
- Complex preparation (per 20 wells)
Tissue culture 96-round bottom well plates were used for complex preparation:
- Prepare 0.02 μg/μl gWIZ™ SEAP DNA solution in OptiMEM® or sterile deionized water.
- Add 6 μl of reagent to 100 μl of DNA solution.
- Mix carefully by pipetting (10-15 times).
- Incubate 5 minute at room temperature.
- 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.
Place the cells into CO2 incubator for 24 hours.
- 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.
- Transfer 5 μl of transfected cells supernatant into a well of flat bottom 96-well plate.
- Add 100 μl of PNPP reagent.
- Incubate for 5 min at room temperature in the dark place.
- Read the plates at 405 nm.