I thawed a 10 years old stock of J774A.1 from liq. N2 and tried to grow in 6cm dish using High glucose DMEM supplemented with 10%FBS, Pen/Str and Normocine. It's been 16 hours to culture but cells are still floating. The plate is almost 80% saturated with the floating cells. Can someone please tell about the attachment time to the plate? Should I throw the plate and use the new stock or wait for some more days? Is there problem in my culturing practice or medium? Please guide me. Thank you.
The floating cells probably are all dead but if there is no contamination you can still continue the culture. I would not recommand to include normocine routinely in the culture medium because it is slightly toxic. Routine screening for mycoplasma contamination is better and Pen/Str usually is enough to prevent bacterial contamination. If it is not you should rather work on the sterile handling. If cells are thawed during these 10 years you may have problems because a lower fraction of them are still viabile. Removing the DMSO used in the freezing solution is important because it impairs cell viability. The use of small culture vessels initially may be helpful for immune cells because they tend to create themselves a favourable environment. Coating of flasks with collagen is a common way to facilitate attachment.
Hope this helps
I also have face the same problem as you. But i use AAV 293 cells and 293tt cells, there are high numbers of floating cell after 24 hrs of culturing. I change my FBS and use some good quality FBS with mycoplasma inhibitor. Its than work for 293tt cells and no floating was observed. However, it didn't work for AAV 293 cells .
So i would recommend, that if your FBS is not of good quality, than you can try with good one and also add mycoplasma inhibitors. I hope it will work
You may try to coat the plate with poly-L-lysine. It helps the cells to attach.
Thank you all for your valuable suggestions. I just noticed that there were some moving particles (contaminants) which might be responsible for no attachment and eventually death. I will follow your suggestions while thawing a new stock.
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