Monocyte Differentiation and Maintenance

THP-1 monocytes (ATCC TIB-202) and primary monocytes were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% heat-inactivated fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO₂. THP-PPM1A cells were generated previously21 (link). Human PBMCs were isolated from buffy coats by the Ficoll-Paque density centrifugation method. Monocytes were enriched by positive selection using anti-CD14 mAb-coated microbeads from Miltenyi Biotec (San Diego, CA) according to manufacturer’s protocol. Monocytes were then differentiated with 5 ng/ml GM-CSF (R&D Systems, Minneapolis, MN) for 5–7 days to obtain monocyte derived macrophages (MDM). fetal bovine serum was obtained from Life Technologies (Grand Island, NY). Phorbol ester 13-phorbol-12-myristate acetate (PMA), puromycin, etoposide, kanamycin and anisomycin were purchased from Sigma (St. Louis, MO). Sanguinarine and SP600125 were purchased from Fisher Scientific (Pittsburgh, PA). CellTiter-Glo luminescent cell viability assay kit was purchased from Promega (Madison, WI). Polyclonal PPM1A antibodies were purchased from Thermo Scientific (Rockford, IL). Monoclonal mouse antibodies to GAPDH and α-tubulin were purchased from Santa Cruz (Dallas, Texas) and Cell Signaling (Danvers, MA), respectively.