Embryoid bodies were cultured as follows. ES cells were treated with trypsin briefly to retain cells in medium sized clumps. Cells were then centrifuged at low speed and resuspended in ESC Growth Medium without LIF and cultured in bacteriological grade petri dishes. ESC Growth Medium without LIF was changed every day for 9 days with minimal disturbance to the embryoid bodies. Samples were taken at specified time points using TriReagent according to manufactures standard protocol or fixed in 10% NBF for histology.
