293T cells: how to sort it the problem of detaching after washing with PBS - (Jul/24/2007 )
I tried to use 293T cells but I found they were so easy to detach after my washing with PBS, which made me lose quite a lot of cells. but there are many protocols need to use PBS washing. how did you sort it out?
We do it like this: take the flask, turn it so that the cells are at the "roof" of the flask, pipet PBS on the side of the flask with no cells on it, then turn slowly to get cells in contact with PBS. Aspirate the PBS off again. We never pipet anything directly on top of these cells.
Your problem is that the your PBS does not contain Ca and/or Mg ions (don't know which one is important, probably Ca). You need a buffer with those, and then the cells will not detach. I don't have a problem if I wash them very briefly in normal PBS. But if I want to leave the solution on for longer than 2 minutes, I'd use a Ca-containing one. You can also try using TBSS or medium without serum for wash. Those contain Ca ions.
stardust is right as well. These cells are very weakly anchored even when they are attached.
If the PBS wash is done for a few seconds, you will not lose cells. But if you to wash several times, use PBS with calcium and magnesium. It can help. and iif you still have a problem, coat the dishes or plates with poly lysine and then seed the cells. This can help cell attachment.