1. We would use serum-free media in the insert, then put serum media in the well. This produces a gradient to that cells are enticed to move through the pores.

2. In my experience, you won't have enough cells to do flow with after they invade through the pores. I suspect they'll just attach to the bottom of the insert rather than float down through the media. You'll find you might have to stain the bottom of the insert with crystal violet, then count the cells, either manually or with some imaging analysis software.

Also, this experiment is what we consider invasion, since they are moving through an ECM in the pores. For migration, we don't use the transwell inserts, but rather stoppers like this kit from Enzo: http://www.enzolifesciences.com/ENZ-KIT113/cell-migration-assay-tc/

Since here the cells aren't moving through an ECM, just moving across the culture surface, we considered that migration. Here, you could just stain nuclei then count the number that have moved into the stopper area after some timeline, say 16-24 hours.

3. I can't say what the macrophages will do! You might have to give it a whirl and see what they decide is best for them. ;)

Thank you so much Seron! This is very helpful.

One quick question: do you think its possible to collect the insert, dip the bottom in trypsin and then collect the cells for flow? Or would you still recommend staining with crystal violet?

You can certainly collect the cells for flow. We are collecting them as a way to create invasive clonal populations that have been transformed from normal cells so that we can characterize them. From this, we get new cell "lines" to work with, so flow shouldn't be too big an obstacle.

The only issue I see is that you might not have enough cells to do flow. We don't get lots of invasive cells, but if you do get lots of them, you could probably get enough over a few inserts  to do a flow experiment. Most protocols I've worked with for flow have you start with a million cells for fixing/staining. Seems like a tall order for inserts, but you can always scale it depending on how many you get per insert, or use larger inserts. We've always just used 24 well ones. I'm sure there are 6 well inserts. Good luck! 

i have a question regarding J 744 A1 macrophage cell line, i am trying to do transwell migration assay using 8um pore with out any ECM, is it true that these cell will adhere to inner side of the filter and not float in lower chamber? is coating the outer part with ECM necessary?

if they adhere to the inner side of surface how can i count them?

Hi Manasi, have you been able to optimize the protocol for macrophage migration and invasion? I am starting out on macrophage study and would like to know how well the protocols work.