Hy everyone. We are running migration and invasion assays with U2OS cells following basic protocols from literature. We have used HGC-27 cells and it works fine but with U2OS cells we see very little migration. We are using 10e4 to 10e5 cells in the upper side of 8uM pore size chambers with serumm free medium. We have also starved the cells for 24 hours. In the botton chamber we´ve tried 10% and even 30% serum as inducers of migration. In any case it didn´t work.
Interestingly I saw a paper describing the protocol like this "About 1 × 104 cells in DMEM containing 10 % FBS media were added into the top chamber, and the bottom chamber was filled with FBS-free DMEM". Is this correct?
So I ask, are there some tricks we are missing?
Thanks
I will suggest you to put cells in the upper chamber with 1% FBS and in lower chamber with 10 % FBS. According to my experience, many types of cancer cells even die without FBS. hence no migration will be seen in such case.
Are you coating the filters? it could also be that your cells have problem in attaching to the filter. I never used those cells, but being an osteosarcoma I expect a good expression of alphav-beta3 integrin, so I would give a try with vitronectin.
In alternative, adding a little amount of serum in the top chamber as suggested above, it could help by depositing some of the serum proteins on to the filter (assuming you are not coating/blocking it).
hope this help,
Good luck
Max
How long are you leaving the cells in upper chamber for the assay?
Maybe you need to change the time.
Thanks for the answers. I will try having some FBS in the upper chamber. W ehave left them for 24 hours.
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