In particular I have some problems about aggregation at first step: the cells become "dark" after 2 hours. When I used the same protocol with mouse ESC I didn't have any problems.
In my personal experience I find that hESCs don't aggregate as a single embryoid body in either u-bottom 96-well, but do so in v-bottom, but only some cells successfully sink to form the EB (the rest remains floating). I use a v-bottom and centrifuge at 200 xg for 1 min to let the cells aggregate.
Make sure you remember to add 20 uM of Y-27632 (ROCK Inhibitor) to your cell suspension if not your cells will die when dissociated.
If you are worried that the dark cells means they are dead, you can always extract from 1 well and do a Trypan blue stain to check.
This is Sasai's protocol that uses hESCs for SFEBq, they use v-bottom wells (though for optic cup formation and not hypothalamus)
http://www.cell.com/cell-stem-cell/abstract/S1934-5909(12)00242-1
By the way there is an alternative protocol that does a similar thing by Lancaster:
http://www.nature.com/nprot/journal/v9/n10/full/nprot.2014.158.html
I have never tried V-bottom, I am a doubt :is it easy to pick up the SFEBq? Now I used Tryple instead Accutase with 50uM Rock to do a single cell and the cell survive. Thanks for references the second I don't know.
Good to hear that your cells are surviving now. To dislodge the EB, I just use a 1 mL pipette to pipette up a portion of the media, then release it quickly into the well, the EB will get dislodged easily.
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