Vitronectin coated polystyrene tissue culture plates

H9 hESCs were initially seeded as colonies on 3.5 cm 2 vitronectin-coated polystyrene tissue culture plates (Sarstedt) and expanded in E8 ® media (Stemcell Technologies). Cells were cultured and passaged upon reaching approximately 70% confluency. Cells were washed with PBS, released from the plates using 1 mL of ReLeSR ® (Stemcell Technologies), and resuspended by mechanical agitation. Finally, 50 µL of cell suspension was added to a fresh vitronectin-coated plate, and allowed to grow for 5 -6 days until reaching approximately 70% confluency, while replenishing with fresh E8 ® media every 1-2 days. NIH 3T3 cells were initially seeded on 3.5 cm 2 polystyrene tissue culture plates and expanded in DMEM ® + GlutaMAX TM (Gibco), supplemented with 10%v/v One Shot TM Fetal Bovine Serum (Gibco) and 100 U/mL penicillin-streptomycin (Gibco). Cells were passaged upon reaching approximately 70% confluency, the cells were washed with PBS and released from the plates using 500 µL of TrypLE TM (Gibco), incubated at 37 o C and centrifuged at 300x g for 5 min. Subsequently, the supernatant was removed and the cell pellet was resuspended in 1 mL of DMEM ® + GlutaMAX TM . Finally, the cells were passed at a 1:10 split ratio and allowed to grow for 3 -4 days until reaching approximately 70% confluency, while changing the media every 2 days.