Hi all
I am working on immortalized human keratinocytes N/TERT-1 cells and trying to produce KI and KO cell lines by CRISPR/CAS9 system. i successfully got the single clones but when i tried to split these clones from 48 well plate to 12 well eventually all the cells died. i don't know whats wrong with these cells. as these cells are very fragile to shearing force i usually flick it with finger and genlty pipette up and down. there are following conditons of the passaging
wash with PBS, add 0.25% trypsin (80 ul in 48 well plate), incubate for 6 minutes, add 80ul DMEM, mix genlty, than centrifuge 500g for 6 minutes, than discard the media and flick with finger to mix the cells , than add SFM Keratinocytes media , genlty mix than add in 12 well plate.
If someone has an experience of working with these cells please help me out about the situation.
Thanks
Some general protocol stuff first:
- is there serum in your DMEM you use to neutralize the trypsin? If not, your trypsin is still active.
I work with primary keratinocytes, but they are generally very sensitive to lots of things, but especially viral infection and low confluence (both of which you are doing).
Sometimes, giving the cells a long time (7-10 days) after that final split can yield colonies. Try giving them some time.
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