Whole blood cells or PBMC were labeled using the following monoclonal antibodies: CD3-PC5 (UCHT1, Beckman Coulter-BC, Paris, France), CD4-FITC (13B8.2, BC), CD8-FITC (B9.11, BC), CD28-PE (B-T3, Abcam, Cambridge, UK), CD25-PE (M-A251, Becton Dickinson-BD Biosciences-Pharmigen, San Diego, CA, USA), CD56-PE (N901, BC), HLA-G-PE (MEM-G/9, Abcam), and PE-Cy5 Conjugated anti-human Foxp3 (PCH101, eBiosciences, San Diego, CA, USA). All procedures were performed according to the manufacturers’ instructions. For intracellular staining of Foxp3, the cells were labeled with CD4-FITC and CD25-PE antibodies and were stained intracellularly for Foxp3, after fixation and permeabilization. Whole blood samples were treated with BD Pharm Lyse buffer 1× (BD) for 15 min, after staining, to lyse red blood cells.
Flow cytometry was performed on an EPICS Coulter XL-MCL Flow Cytometer (BC). At least 20,000 events were acquired for extracellular and 100,000 events for intracellular staining. Data analysis was performed using the FlowJo V7.5 software (Tree Star Inc., Ashland, OR, USA).
Flow cytometry was performed on an EPICS Coulter XL-MCL Flow Cytometer (BC). At least 20,000 events were acquired for extracellular and 100,000 events for intracellular staining. Data analysis was performed using the FlowJo V7.5 software (Tree Star Inc., Ashland, OR, USA).