Hello, I recently recieved a frozen batch of Dox inducible HeLa cells to generate protein of our intetrest. I have passaged the cells in DMEM with 10% FBS and 1% Penstrep for almost 3 weeks now but my cells still look rounded rather than have their characteristic morphology. I have also increased the serum percentage to 20% with no difference in results. Could someone please suggest a solution. Thank you
Cheers
Pranetha
Hi Pranetha. In my faculty, we have found that not all culture plasticwares allow cell adherence. My cells were either rounded or floating when I used certain 6-wells brands. I would suggest you to transfer the cells immediately into a new cell culture based palsticware, best to use Corning Costar. Please keep us posted on your updates.
Hi Kerry
They are adherent cells. I am growing them in 75cm2 NUNC culture flasks.
Thanks for the suggestion Nurul. Will try plating them in a corning dish and see if they are any better.
Hi,
Agree with above comments. Apart from that It could be the trypsin. You need to be very careful about removing the trypsin from the cell suspension before reseeding. If you leave the trypsin in the medium, it may not be fully neutralized by the added medium and could damage the cells. This phenomenon seems to be worse with some cell lines and gets worse as time goes on. Try HyQ-Tase from HyClone (Thermo now). It's gentler on the cells, can be left on for longer than usual without harm, and is inactivated after about an hour at 37°C.
If you do use trypsin, you might try following this protocol from ATCC for these cells: Volumes used in this protocol are for 75 sq cm flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Note: if floating cells are present, it is recommended that they be transferred at the first two (2) subcultures as described below. It is not necessary to transfer floating cells for subsequent subcultures.
Hope this helps
Hello
i agree with all researchers . and in general this cell culture with MEM or DMEM with Earle's Salts +10% FCS + 2mM L-glutamine + non-essential amino acids, or RPMI-1640 with 5% FBS
in pdf protocol you can help you
Good Luck
This is a Tet-On cell line, needs a special FCS confirmed as tetracycline free. You did not mention this. Try to pick up some to save your line if don't use that type.
Thank you for all the help. I used Corning plates and the cells look fine now.
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