Annexin V/PI Apoptosis Assay
Cell apoptosis assay was performed as described previously [51] using the Annexin V–FITC apoptosis detection kit (BD PharMingen, San Diego, CA). Briefly, human monocytes (5×106) were incubated with inhibitors for 30 minutes in X-vivo medium and restimulated with 100 ng/ml M-CSF overnight. The cells were removed from the culture dish using Accutase (eBioscience, San Diego, CA) and stained with Annexin V–FITC and PI and analyzed by flow cytometry (FACSCalibur; BD PharMingen). Annexin V-FITC and PI double negative cells were considered non-apoptotic cells for statistical analysis.