Dear All,
Im working with Ovcar-3 cell lines. When I thaw the new vail the cells are floating, they are not attachng. To make sure there is no contamination, I tried fresh medium, different incubator and Ovcar 3 cells from different source but still cells are floating. Can anyone please suggest me a solution to this problem.
Thanks
Sachin
Which medium are you using? Recommended is RPMI-1640 + 20% FBS.
Check your cells for mycoplasma contamination.
Hi Sachin, assuming that you are using the correct medium (RPMI1640 + Serum) and flasks/dishes with the right coating for your cells, I would think that your thawing procedure is killing the cells, or that the stock is ruined. To verify this, you could ask someone else with experience using these cells to thaw an aliquot and see if they can get them to grow.
Else, general guidelines for cells thawing are as follows:
Good luck
Are the floating cells viable? Do they exclude trypan blue? If they are not viable, something went wrong during the freezing process, during the thawing process, or during storage in the frozen state.
How long are waiting before checking for attachment? Have you checked the pH of your media?
Thanks guys for the suggestions. The cells were in the constant culturing 15 dyas back and they were growing very well. I froze 15 vails and stoped the culturing for the 7 days. I am trying to grow these cells from last 7 days they were floating even after 24 hours. I autoclaved every thing, used frshed preapred medium, FBS, PS but still they are floating. Please help if anyone have solution to this problem.
Thanks
Sachin
OVCAR3 cells are quite sensitive. i have faced a similar problem before. the probe was in the whole batch of vials i froze then. when i revived the earlier vial it grew well in the same media i tried to culture the more recently frozen vial. i suspected tht problem was in freezin procedure. i guess here is the same problem.
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