Cd8 biotin

Cells were stained with specific antibodies or control isotypes using conventional protocols. The following anti-mouse antibodies were used for FACS analysis of MDSCs: CD11b/PECy5, Gr1/PE, Gr1/APC, CD62L/FITC, F4/80/FITC, CD11c/PE, and MHC CI OVA peptide/Biotin from eBiosciences (San Diego, CA); as well as CD124/PE, CD40/FITC, CD86/FITC, I^A-I^E/FITC, Ly6C/FITC and Ly6G/PE that were purchased from BD Biosciences (San Jose, CA). Lymphocytes characterization was performed with the next specific antibodies: CD8/PE and CD8/Biotin, CD69/PECy5, CD62L/FITC, CD247/PE, CD4/PE, CD4/PECy5, CD25/PECy5, Foxp3/Biotin, IFN-γ/FITC, IL-4/PE and IL-17/PE, all from eBiosciences. FITC-conjugated Streptavidin was purchased also from eBiosciences. In all cases, cells were previously incubated with anti-mouse FcγR 2.4G2 ascites (HB-197; ATCC) to reduce the non-specific binding. For intracellular staining, cells were fixed and permeabilized, according to the manufacturer’s protocol, with either Foxp3 Staining Buffer Set or IC Fixation and 10X Permeabilization Buffers from eBiosciences. Cells were acquired using both FACScan (BD Biosciences) and Gallios (Beckman Coulter, Miami, FL) flow cytometers and analyzed with Kaluza 1.2 (Beckman Coulter) and FlowJo 5.7.2 (Tree Star Inc., USA) softwares.

For the flow cytometry analyses, cells were stained with monoclonal antibodies against the following anti-mouse molecules: CD3-biotin, Ter119-biotin, GR-1-biotin, B220-biotin, CD11b-biotin, NK1.1-biotin, CD3-biotin, CD4-biotin, CD8-biotin, cKit-BV650, Sca1-PE, CD135-PerCP, CD34-APC, CD25-PE, CD44-APC, CD3-APC, CD8-PerCP, CD-BV650, CD45.1/Ly5.1-PE-Cy7, CD45.2/Ly5.2-APC-Cy7 (all from eBiosciences, CA, USA). For secondary detection of biotinylated antibodies, streptavidin conjugated with FITC or PE-Cy7 was used (eBiosciences, San Diego, CA, USA). All flow cytometry measurements (Canto II BD biosciences or LSRII BS Biosciences) were calibrated first with BD™ CompBead Plus, κ/Negative Control (BSA) Compensation Plus (7.5 µm). Recommended flow cytometry settings are specified in [17 ]. Flow cytometry analyses were performed using FlowJo software (Treestar, Ashland, OR, USA).