- 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®.
- Complex preparation (per 20 wells)
Tissue culture 96-well round bottom plates were used for complex preparation:
- Prepare gWIZ SEAP DNA solution in Opti-MEM®I or sterile deionized water, 0.0125μg/μl.
- Add 160 μl of DNA solution per well.
- Mix carefully by pipetting (10-15 times).
- Incubate 5-10 minute at room temperature.
- 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.
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 mg/ml of PNPP is prepared in1M Diethanolamine, 1mM MgCl2, pH9.8.
- Pipette 10 μl supernatant of transfected cells 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.