THP-1 macrophage polarization, staining and flow cytometry

To generate non-polarized (M0) macrophages, 4x10⁵ THP-1 monocytes were differentiated with PMA for 24 hours, after which the PMA-containing medium was replaced with 10% FBS-containing 1X RPMI growth medium and the cells were allowed to recover for 24 hours.

M0 macrophages were then treated with equivalent amounts of exosomes, based on 30 ug of exosomal protein. After 72 hours, cell culture medium was removed and stored at -80°C for analysis of secreted chemokines and cytokines (see below), and cells were detached using Accutase (Innovative Cell Technologies). After pre-treatment with Fc Block (BD Biosciences), 1x10⁵ cells were incubated for 30 minutes at 4°C with fluorochrome-conjugated primary antibodies against the following proteins: CD14 (ThermoFisher Scientific), HLA-DR (BD Biosciences), CD163 (R&D Systems), CD80 (Bio-Techne), and CD206 (BD Biosciences). Cells were washed twice with FACS buffer (0.8% BSA in PBS), resuspended in 0.5% PFA and stored at 4°C protected from light before being acquired on a LSRFortessa flow cytometer (BD Biosciences). Positive controls for M1 macrophage polarization were 20 ng/mL LPS and 20 ng/mL IFNɣ and for M2 macrophage polarization were 20 ng/mL IL-4 and 20 ng/mL IL-13. M1 and M2 polarization controls were treated for 24 hours. Data from at least 3 independent experiments were analyzed using FlowJo software (TreeStar).