There are many different protocols for THP-1 differentiation to macrophages using PMA.
I am doing the following procedure:
1) seed macrophages at a density of 1x106 cells/mL treated with 50 ng/mL of PMA.
2) after 24h incubation discard the medium and replace with frash medium
3) after the second 24h incubation treat macrophages with different stimuli (for macrophages, M0, treat with fresh medium) and incubate for 24h/48h/72h