Dear Pablo Leal Cardozo ,

I think there are two separate problems to solve. The problem you have with split/subculturing the cells and the actual migration assay.

From my experience with PBMC derived macrophages. They attach so stiff to normal cell culture plastic that you are not able to detach them without killing them at the same time. There are some protocols (i.e. ice cold Trypsin on ice for 15 min) on the internet, but in my opinion they are all bs and have never worked for us. We are using successfully for years costar ultra low attachment plates (6-well). When using these you can spill your macrophages loose with a 1000 µl piepet or use a cell scraper. I really love them - you should give them a try.

This is my second answer on this topic to my knowledge it also worked for max here:

https://www.researchgate.net/post/Is_passaging_of_PBMC-derived_macrophages_feasible

If you want to use them in a migration assay where you do not have to passage them you might want to take a look µ-Slide Chemotaxis from ibidi. They are also tricky and you might get into trouble using such a long culture time, but at least you can plate and differentiate them in there and use them down stream doing an image based migration assay. But you should have access to an life cell microscope with an automatic stage to acquire three images sets from multiple positions at the same time.

Best wishes

Soenke

Thanks for the tip Soenke. I'll look into those plates you suggested!!! Since I have several groups to analyze, I'd want to give another shot to the transwell system, before moving to the Chemotaxis chamber, since I can read several analytes at once using this assay.

If it doesn't work, I'll prioritize my targets and move to the chemotaxis chambers, instead