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Cells do not attach in the flasks - (Oct/11/2008 )

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Keeping cells in the -70 for long term does not work, apparently they very slowly metabolise and the DMSO damages the cell membranes, killing the cells. I have successfully thawed cells that have been in the -70 for a year, but they were very very unhappy. 2 vials of A549 (grow very quickly: population doubles every 12-18 hours normally) that I would normally seed into a T75 each were seeded into a single well of a 6 well plate to give me enough cells to continue the culture.

If you want to keep cells long term, store them in a liquid nitrogen dewar.


While I agree that -70º is not the best place for cells, I have been in labs where they kept HepG2 cells in the -70 for over a year. They would thaw a new aliquot once in a while and change their cells.

Now, back to your cells.
I understand that you can`t give any details, as it is likely there might be intellectual property and/or patenting issues. So you need to think if your RNAi could interfere with the cytoskeleton or with cell adhesion properties. But, did you ever see something like this when you first raised your cells? Are you sure they are still alive? You might want to get hold of some Matri-gel (look in a neuro-lab, do not buy a whole bunch. It`s expensive and your might never need it again). Thaw the matrigel in ice IN the refrigerator (4ºC), dilute 1/3 with COLD medium and add your cells. Plate and watch it polymerize. If your cells do not attach to that, well, then I am truly out of ideas.

Matrigel is tricky, I have used chilled down pipettes (tips, plastic-ware, etc) in order to keep it from clumping.


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