Hi Roberta, I have no experience in culturing A549 cells, however I performed it using B lymphocytes which are pretty much smaller than A549 cells, I bet. The pore size of transwell I utilized was 8 um. Therefore I suspect that 3 um might not work regarding your case. Anyway you can try it for one time to see the whether it works or not.

Hope it is helpful for you. Good luck! 

Do you treat A549 with some drugs? Because after treatment they might have changed  the morphology (enlarged, flattened) and thereof pores could be to small for them to go through. I have always used the scratch assay/wound healing assay instead. Cheap, quick and no limitations in the cell size.

8 micron is the generally accepted pore size for cancer cells!  3 is most likely too small, you should use one of the smaller inserts with 8 available.

Steve

Thank you all for your answers! I found that also 5 micron transwells work for these cells. I should try with 3 um. 

Agnieszka, no treatment with drugs. I cannot use wound healing assay instead, because I need to recover migrated cells.