The cell is dying with unknown reasons.

Hi:

If they attach/expand in the flask, this means you have viable cells in the first place but something happens during the culture. I think you should consider a new batch of your culture medium (DMEM probably?) or FCS. Some cell lines may not tolerate antibiotics and are recommended to be cultured in antibiotic free medium. A low possibility for Mycoplasma contamination, which should be ruled out with PCR.

Good luck

Dear Bashir,

After 12-16 hrs of revival change the medium, it will remove all dead cells.

Culture should not be over confluent. Split the cells after 70-80% confluency. It will help.

Thanks

I agree with Sagar. Replacement of media (I used RPMI+10% FBS+pen/strep) next day after I thawing may help. Another potential issue might be DMSO (usually added during freezing of cells). Did you wash cells with media prior toplating them?

Hi all,

Thanks a lot for these important informations.

But I did the same as Sagar and Rinat said, I changed the media (RPMI 1640+10% FBS+ Antiboitic) after 24hr, and I have check the contamination (no contamination was found), bur when I check it after another 24hrs, I found most of cell floatng, and after 48hr almost all cell die.

so I don't know what the problem exactly?

First of all, you should use an antibiotic-free medium (all types of antibiotics are a bit hard on cells so don't use it until you get your cells up running). Secondly, I'm a bit scared on the way that you're thawing your cells. To thaw your cells, get a 15mL tube filled with media (approx. 13mL), then bring a beaker with a 37oC warm water and dip the cell vial 4-5 times repeatedly. Once cells are thawed, quickly transfer them to the tube for centrifugation. Do 5 minutes at 1200 RPM. Decant supernatant and re-suspend your cells with the antibiotic-free media and add it to an appropriate flask. It is very important that you know how many cells were stored down so you put them in a suitable flask. You don't want to put many cells in a T25 or very small number of cells in a T75. For your 500mL medium, add 50mL FCS or FBS + 5mL L-Glutamine + 5mL Na pyruvate. Make sure to come the second day and change the media with one wash using the your media (so take off the old media + add some fresh media to wash then take it off + then add your media). I don't usually do this washing step but I want you to do it because you're experiencing this problem.

All the times floating cells are not dead cells. It depends on characteristics of the particular cell line. Confirm the floating cells are live/dead by simple trypan blue staining.

collect all the floating cells (after 48hr) and seed them in fresh flask.