Hello,
I have an suddenly and recently happened problem of HaCaT cell line after splitting step.
I use the same 175T flask and 100 mm dish, fresh DMEM medium, FBS, TE buffer, but I have never observed this kind of problem for last 1 year with HaCaT cell line. And I also culture other cells (3T3-L1, B16F10) without any problems with those stuffs and subculture protocol. I already discarded the cells and re-thawing other HaCaT vial from N2 tank, but the problem repeated.
Can you recommend any method to fix it?
Thank you.
Hello, If it worked well before in similar conditions that you are using now, then something must have changed. Factors to consider: passage number of the cells you use, initial seeding number, tissue culture coating of the flask. How does the confluency look and the timeline of it ?
Try splitting them into a new flask or a new dish.
alternatively, maybe something happened to the incubator (no CO2, wrong temp, etc) which affected adhesions/viability.
Cheers
Please describe your splitting step. Do you use trypsin or another enzyme dissociative? Are you attempting to put them back into a flask or a dish that has been previously trypsin treated? If so, trypsin can disrupt the tissue culture coating of plastic ware and this is why a new flask/dish must be used after trypsinizing/enzyme treating.
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