hi, we have tried this protocol several times but the cells...

Thank you!

No need for more matrigel. The matrigel is only needed very early to promote polarization and trigger the right developmental program.

Thank you so much for the reply! We use E14 cell line. The EBs seem develop well on Day1 but we do not see clear edge on Day3. Then the EBs became darker and on Day7 we do not see any live cells. If we move the EBs to 6 well plate on Day4, do we need to add matrigel in the medium?

I thought perhaps I should further clarify: if (a) your EBs develop well during the first few days (i.e. on Day 1 you see a well-defined ball of cells with smooth edges and on day 3 there is a clear epithelium developing all around the EB) and the media is changing color to yellowish before day 7, I would move the cells earlier to the 6 well plates. If (b) the EBs don't form well, they become darker, you don't see smooth edges from day1 or the neuroepithelium is not clearly developing from day3, then probably your dissociation is too aggressive and I would try to reduce the time for the enzymatic dissociation.

Hi Min,
You do NOT change media during the first few days. When are your cells dying? and also, what cell line are you using? you could try to move them to the bigger plates earlier (day 4-6) if your cell line tends to grow very quickly and that might be the issue. I hope this helps but don't hesitate to email me if you need any further info. Good luck!