Fibronectin coated 96 well culture dish

The following methods were used to culture and proliferate PB-SSEA-3(+) cells: (1) ∼10 × 10³ cells were plated onto a 96 well-culture dish per well (Corning) and cultured in DMEM with 10% FBS at 37°C, 5% CO₂. For tracing, cells were labeled with Hoechst 33342 and vibrant CFDA-SE cell tracer (ThermoFisher). (2) ∼10 × 10³ cells were plated onto a fibronectin-coated 96 well-culture dish per well (Corning) and cultured in DMEM with 10% human serum (pool of donors; BioIVT, Westbury, NY, USA). (3)∼10 × 10³ cells were plated into each well of a fibronectin-coated 96 well-culture dish (Corning) and cultured in DMEM with 5% FBS and 5% inter-α-inhibitor, known to promote attachment and long-term growth of pluripotent stem cells such as embryonic stem cells by activating Yes/YAP/TEAD transcription pathway¹⁸ (Athens Research And Technology, Inc., Athens, GA, USA). (4) MethoCult (H4100; STEMCELL Technologies) was diluted in 20% (vol/vol) FBS plus α-minimal essential medium to a final concentration of 0.9%. MethoCult (100 μl) was placed into each well of a 96-well plate (Corning), ∼10 × 10³ cells were seeded on the top of MethoCult, and then another 50 µl MethoCult was added. The cells and MethoCult were mixed thoroughly by gentle pipetting and cultured at 37°C, 5% CO₂. To change the medium, 10% of the volume of the initial MethoCult culture medium was gently added to the wells every 3 d.