I transferred KB cells stored in -80 into a T-75 flask by adding 10% FBS media without centrifugation.
I plan to change the media after proper adhesion of the cells. I don't know how many days it will take the cells to adhere and when to subculture it. Please let me know how many days it would take those cells to be revived. And also when to subculture it
Hello V H Krishna Prasad
It takes approximately 1-2 hours for strongly adherent cells but may take 7-8 hours for less adherent cell types to adhere to the substratum You may monitor adherence using a microscope.
In case if cells fail to adhere to the substratum, it indicates that the cells are under stress. There are many factors that could cause stress in culture.
1. Have you thawed the cells in a proper way? (quick thawing)
2. Temperature fluctuation in -80 freezer during storage of cells.
3. Incubator temperature fluctuations.
4. Inappropriate environmental gas mixture.
5. Contamination in culture.
6. Insufficient or inappropriate cell culture substratum.
7. Inadequate nutrients in cell culture media.
For a good culture, it should not take more than 48 hrs. to revive in a stress-free environment. Once they grow well in culture, the cells should be sub-cultured when the culture is 80% confluent.
After thawing, if you have not centrifuged the cell suspension to get rid of DMSO, the media should be changed once the cells have attached to the substratum or in the early hours of the following day.
Best.
Thank you for your input Sonja Eberth, née Röhrs
I changed the media after 24hrs. Since the cells are already under much stress due to preservation I thought it would better to skip separation of DMSO for the time being. I am surprised to notice that many cells have survived and adhered compared to the previous time where I had centrifuged them to separate DMSO. Please have a look at the attachment fileSonja Eberth, née Röhrs
. Also kindly elaborate why KB cells cannot be used ? They are used as oral cancer model- Badges
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