Embryoid bodies generation
Add Trypsin 2ml and gently shake plate.
Add 8ml ES medium without LIF to inactivate trypsin.
Centrifuge at 500rpm for 10min.
Add 8ml of EB medium to each 10cm petri-dish while centrifuging.
Gently resuspend pellet in 5ml EB medium by 5ml pipette, up and down for 5 times.
Take 100ul cell suspension and dilute into 1ml.
Count.
Take appropriate number of cells and dilute into 1×10^6 cells/ml.
Gently re-suspend by 5ml pipette, up and down for 4 times.
Seed 2ml of cells/dish.
Save 2×10^6 cells for RNA extraction.
Transfer medium containing Ebs to 50ml tubes.
Carefully add 5ml medium to dishes immediately and put back in incubator.
Sink cells in tubes by gravity for 5min.
Discard supernatant.
Add 5ml medium to pellets.
Mix samples by inverting a few times gently.
Distribute samples to different dishes.
Prepare gelatin coated chamber slides (2 well).
Add 1.5ml of medium into each well.
Transfer 1 dish of day4 Ebs into 15ml tube.
Wait for Ebs sink to the bottom of tube by gravity for 5min.
Discard supernatant.
Add 10ml EB medium into pellets.
Seed 0.5ml of EB to each well.
Change medium everyday: suck supernatant, replace with fresh EB medium.
Take 2 wells for each group on day6, day8 and day12.
Suck the medium carefully.
Fix with 4% PFA for 30min @RT.
(in hood).
Wash #1 with 2ml of PBS.
Wash #2 with 2ml of PBS.
Wash #3 with 2ml of PBS.
Store samples in PBS, @ 4°C.
