I tried to thaw several stocks from the -80 freezer that date back to one year and others from liquid nitrogen that date back to two years but they die continuously. I thaw them in 6-cm culture dishes but they are very weak and very slow. I waited for so long until one plate became confluent and then splitted it in two 10-cm plates. When they were passaged they became even weaker and every day I look at them, I find a lot of floating cells and nothing improves when I change the medium. N.B. I culture them in DMEM with 10% FBS.
I'd start by looking for contamination - look for mycoplasma, small bacteria, etc. That might be hurting your cells.
Alternately, try and find the same cells from a different researcher, or order a new stock from ATCC. It's possible that something just went wrong in freezing them down, and that they're simply no longer healthy enough to survive.
Hi Aya,
Did you rinse the cells when you thaw them to eliminate the DMSO or cryopreservative medium?
What I use to do: Resuspend the thawed cells (vial of 1-1.5ml) as quick as possible in 10 ml of medium (DMEM with 10% FBS). Centrifugation as usual, throw supernatant, resuspension in fresh medium (DMEM with 10% FBS) and put them in culture dishes. But as William said, maybe it's due to the freezing and it's better to start with a new stock. I think that the B16F10 are not fragile so it should be easy to thaw them.
Good luck!
Dear morgane and William,
thank you very much for your replies. B16f10 used to be not fragile. I used to thaw similar stocks directl in 75cm2 culture flasks without rinsing and they used to be perfectly growing but they are no longer the same. So I started to culture them in smaller plates, wait for longer time but in vain. I also see no signs of contamination in any of the dishes so it remains unexplained to me!
I dont know if you already have found the reason. One problem may be slow thawing. You need to thaw the frozen vial rapidly (2 min).
In addition to all replies above, new stock of FBS might be the culprit. Some FBS stocks don't seem to work for some cell lines. 2.
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