How to prevent EB becoming too large - (Jun/16/2011 )

Hi,

I am using mouse ES cells to form floating EB for 8 days to promote neuronal differentiation (Following 2004 Nature Neuroscience Paper, Volume 7 Number 9 Page 1003). My problem is that the floating EB, after 6-8 days in 10 cm petri dish in floating formation, become large and very dense in the center, presumably starting to die.
I do not have shaker in the incubator...Is there a good way to prevent EB becoming too big? Low density? Or shall I split the cells into multiple dishes?

attach EBs on fibronectin or gelatinized plates on day8. good luck

funny my prob is the opposite. I was tryin to form EBs to differentiate them to hematopoeitic cells but they were too small!

This is what I did:

I used the round bottom low cell bindin 96 well plate. i seeded 1000 cells in 1row and 3000 cells in the 2nd row. after 2 days, i added 80uL of EB medium. 3 days later i changed the medium and 2 days later, I checked and the result was, theye were too small to be used for differentiation.

I was using mouse ipsc

what did u do?