Generation of Bone Marrow-Derived Macrophages

Bone marrow derived macrophages were generated as described38 (link). Human PBMCs were isolated by from whole blood of healthy volunteers by density gradient centrifugation. Lysis of red blood cells was performed using red blood cell lysis buffer (Sigma). Experiments in PBMCs and macrophages were carried out at a cell density of 2×10⁶ cells / ml. All primary cells and cell lines except THP-1 cells were cultured in DMEM supplemented with L-glutamine, ciprofloxacin (Cellgro, Manassas, VA) and 10% fetal calf serum (Hyclone, Logan, UT). THP-1 cells were cultured in RPMI supplemented with 10% fetal calf serum (Hyclone), L-glutamine, sodium pyruvate (Cellgro), and ciprofloxacin. One day prior to stimulation, THP-1 cells were differentiated using 0.5 µM PMA for three hours, washed three times and plated for stimulation. All experiments that were performed for Western blot analysis were carried out in serum free DMEM medium. ATP stimulations were carried out at 5 mM one hour prior to harvesting supernatants.