Characterization of mCD1d-α-GalCer Tetramer

For characterization of the mCD1d–α-GalCer tetramer, cells isolated from various organs were resuspended in PBS staining buffer containing 2% BSA and 0.02% NaN₃. Cells were incubated for 15 min at 4°C with the blocking 2.4G2 anti-FcγR mAb and neutravidin (Molecular Probes) in a twofold excess of the neutravidin–PE contained in the amount of tetramer to be used for the staining. Neutravidin blocking was done to avoid any nonspecific binding of the neutravidin to biotin on the cells. Staining of FITC-, PE-, Cy-Chrome–, and allophycocyanin-conjugated mAbs was done simultaneously with the tetramer in PBS staining buffer at 23°C (room temperature) for 20 min. Cells were washed twice, and staining with biotinylated mAb was performed at 4°C for 20 min. After washing cells, tricolor-conjugated streptavidin was added as a secondary staining reagent for the biotinylated mAb and incubated for 15 min at 4°C, and cells were washed two times before analysis. mAbs used in this study include FITC-, Cy-Chrome–, or allophycocyanin-labeled anti–TCR-β clone H57-597, biotinylated or PE-labeled anti-NK1.1 clone PK136, Cy-Chrome–labeled anti-CD4 clone RM4-4, allophycocyanin-labeled anti-CD8α clone 53-6.7, FITC-labeled anti-CD5 clone 53-7.3, FITC-labeled anti-CD44 clone IM7, biotinylated anti-CD69 clone H1.2F3, FITC-labeled anti-Vβ2 clone B20.6, FITC-labeled anti-Vβ7 clone TR310, FITC-labeled anti-Vβ8.1/8.2 clone MR5-2, and FITC-labeled anti-Vβ12 clone MR11-1 (PharMingen). For intracellular staining, cells were incubated with blocking 2.4G2 anti-FcγR mAb and neutravidin (Molecular Probes) and then surface stained with TCR-β–FITC and either tetramer or anti-NK1.1–PE at 23°C. Cells were permeabilized using Cytofix/Cytoperm Plus™ (PharMingen) and stained using either FITC-labeled anti–IL-4 clone BVD4-1D11 or FITC-labeled anti–IFN-γ clone XMG1.2 (PharMingen) according to the manufacturer's protocol.