Monocytic cells were seeded in 24 well plate at 0.5x10 5 cell/ml in serum free media overnight. Cells were treated with nSMase inhibitor GW4869 (10uM/ml) for one hour or 0.01% DMSO (vehicle) then subjected to stimulation with TNF-α (10ng/ml) or BSA (vehicle) for 6 hours. Monocytic cells (1x10 6 cells) were resuspended in FACS staining buffer (BD Biosciences) and blocked with human IgG (Sigma; 20μg) for 30 minutes on ice. Cells were washed and resuspended in 100 ul of FACS buffer and incubated with CD11c
(S_HCL-3)-PE (cat# 347637; BD Biosciences) or CD11c PE-Cy7 (cat # 117317; BD Biosciences ) on ice for 30 minutes. Cells were washed three times with FACS buffer and resuspended in 2% paraformaldehyde.
Cells were centrifuged and resuspended in FACS buffer for FACS analysis (FACSCanto II; BD Bioscience, San Jose, USA). FACS data analysis was performed using BD FACSDiva TM Software 8 (BD Biosciences, San Jose, USA).
(S_HCL-3)-PE (cat# 347637; BD Biosciences) or CD11c PE-Cy7 (cat # 117317; BD Biosciences ) on ice for 30 minutes. Cells were washed three times with FACS buffer and resuspended in 2% paraformaldehyde.
Cells were centrifuged and resuspended in FACS buffer for FACS analysis (FACSCanto II; BD Bioscience, San Jose, USA). FACS data analysis was performed using BD FACSDiva TM Software 8 (BD Biosciences, San Jose, USA).