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
