While working with differentiated macrophages, it is always recommended to seed the cells before differentiation as lifting the cells once after differentiation is technically difficult and unhealthy procedure considering the adhesion property of cells. Trying to lift the cells after differentiation will result in cell breaking leading to cell loss. Moreover, this procedure may add stress which will be reflected on the experimental outcomes, especially when trying to study M1 and M2 related outcomes.

While seeding monocytes, those unattached monocytes could be dead or worn-out cells. All healthy monocytes do adhere to the surface.

I agree with the above - it's better to seed & differentiate the macrophages directly in the e.g. assay plates in order to avoid detaching them.

Macrophages are strongly adherent and are not effectively dislodged from culture surfaces by standard enzymatic cell dissociation reagents. Indeed, these cells are highly sensitive to improper dissociation procedures.

If you really need to detach and re-seed your macrophages, you try the PromoCell Macrophage Detachment Solution (cat. #C-41330), which is chemically defined and non-enzymatic:

https://promocell.com/product/macrophage-detachment-solution-xf/

It was especially designed for the gentle release of adherent macrophages and guarantees the best possible cell viability, even after prolonged exposure times. Unlike enzyme-based solutions, the PromoCell Macrophage Detachment Solution does not alter cell surface proteins and neutralization is not necessary.