Protocol for STO Cell Transfection by FuGENE HD
STO cells were seeded the day before transfection with the density 15,000 cells per well in 100 μl complete growth medium DMEM + 10 % Fetal Bovine Serum .
Prepare 0.02μg / μl pCMVβ plasmid DNA solution in OptiMEM® .
Add 6μl of reagent to 100 μl of OptiMEM® / DNA solution .
Mix carefully by pipetting ( 10 - 15 times ) .
Incubate 5 min at room temperature .
Add 5μl complex per well to the cells , and mix thoroughly .
Place the cells into CO2 incubator for 26 - 28 hours .
Remove the medium from the well and wash the cells once with 100μl per well PBS .
Fix the cells in the well with 50μl solution of 4 % formaldehyde in PBS for 5min at room temperature .
Wash each well with 100μl PBS .
( 1 / 2 ) Add 50μl per well of substrate / stain solution and incubate the plate overnight at 37°C .
Observe the cells under microscope and evaluate the proportion of blue ( β - gal - positive ) cells .
Wash each well with 100μl PBS .
( 2 / 2 )
