Cd8 efluor 450

T cells were washed in staining buffer (SB) consisting of phosphate-buffered saline (PBS) containing 0.1% human serum albumin (HSA) and 0.1% sodium azide before staining for 20 min at RT. The cells were then washed in SB and fixed in SB containing 1% paraformaldehyd. For intracellular staining, T cells were stimulated for 6 h or overnight with APCs, loaded or not with p573, at a T-cell to target ratio of 1:2 and in the presence of BD GolgiPlug and BD Golgistop at a 1/1,000 dilution. Cells were stained both extracellular and intracellular using the PerFix-nc kit according to the manufacturer's instructions (Beckman Coulter Inc., USA). The following antibodies were used: Vβ3- FITC (Beckman Coulter-Immunotech SAS, France), CD3-eFluor450, CD4-eFluor 450, CD4-PE-Cy7, CD8-APC, CD8-eFluor 450, CD8-PE-Cy7, CD56-PE-Cy5.5 (BD Biosciences, USA) and CD107a-PE-Cy5 (BD Biosciences, USA), CXCR2-PE, IFNγ-FITC, IL-2-APC, TNF-α-PE (BD Biosciences, USA), CD261/TRAIL-R4-PE (BD Biosciences, USA). MART-1 (aa 26–35)-specific TCR was detected with dextramer staining (Immudex) following the manufacturer's recommendations. All antibodies were purchased from eBioscience, except where noted. Cells were acquired on a BD LSR II flow cytometer and the data analyzed using FlowJo software (Treestar Inc.).