Both enhanced green fluorescent protein (GFP) and and improved red fluorescent protein (RFP) called tandem dimer (td) Tomato, also cDNA3.1-based plasmid expressing tdTomato RFP reporter, containing a neomycin resistance gene controlled by the SV40 promoter were used.
The circular plasmid (5 µg) was transfected into about 2 mllion hES cells using Amaxa Mouse ES solution and program A-23 (Nucleofector II). Treated cells were plated into 3 wells of HAFI/Wnt3a feeder cells (in a 6-well plate, after 40 GY irradiation). Two days after, 100 µg/ml G418 were added. Transfected cells were passaged onto fresh HAFI/Wnt3a cells as needed.