Hey all
I recently revived a vial of adherent oral cancer cells and plated them into a T-75 flask. I could notice so many dead/non-adherent cells floating in the media. I thought this would stop after two subcultures, and I would get a homogenous cell in the same cell cycle phase. However, that is not the scenario. I see so many dead/nonadherent cells.
Kindly help
Have you measured the number of live/dead cells? If there are mainly non-adherent cells, this can be explained by tumour cell heterogeneity and tendency to grow in suspension.
Hello V H Krishna Prasad
The cells may not have been frozen properly as per the standard protocol which may have resulted in stressed unhealthy cells. You could possibly revive the culture by centrifuging the cell suspension at 1000rpm for 5 mins, discard the supernatant, tap the pellet gently and add 1-2ml of culture media to the pellet. Mix gently and add to the well of a 6-well plate. Maybe a reduced surface area may help bring a few viable cells in close proximity and your culture may revive. Hope this will work!
Whenever you thaw a frozen vial from liquid nitrogen, always use a smaller flask (like T-25cm^2) for suspending the thawed cells. T-75cm^2 flask may be used for subculturing after the cells have revived and look healthy.
Best.
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