I use dispase to dissociate ips cells, and put it about 1h in the 37oc incubator. the cells are still attached in the bottom of the plate even pipette the cells up and down several times. Is anyone having the same problem?
the Versene I used only dissociate the center of the ips colony, but not the edge of the colony. Does any one any the same problem?
I am frustrated now since so many colonies I need working on
Thanks for help!
XH
Feeder-free or feeder based?
Usually dispase works in my hands within 30 min.
But I remember one case when the dispase didn't work! Still don't know why...
Nowadays I am working feeder-free and use EDTA in a very low concentration.
Cheers
feeder based.
Does EDTA can work on Feeder based culture? What concentration do you use for feeder free culture?
Thanks for your reply.
Qing
EDTA I am using 0.5 mM in PBS. This is working good in Essential8 conditions.
I never ever tried it in feeder-conditions since I moved all my iPSC to feeder-free conditions.
Feeder-based I am only familiar with Collagenase treatment.
1ml collagenase IV (1mg/ml) for 5-15 min at 37 degree. When the edges detach the treatment was long enough. I mechanically cut the colonies in small pieces etc.
Was a lot of work, nowadays my new procedure saves me at least 2/3 of my time!
BEst
Could you ask to Jonas some details? Does EDTA form clamps or single cells? Do you plate the cells in high concentration after this treatment? Is essential E8 medium?Because I always use mTesr1 from StemCell and now I discover this other medium....thanks
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