Anti cd127 biotin clone a7r34

DO11.10 T cells were detected by staining with KJ1–26 TCR clonotypic Ab (eBioscience), and anti-CD4 mAb (clones GK1.5 or RM4–5, BD or eBioscience), after blocking Fc receptors with anti-CD16/CD32. Directly conjugated mAbs were used to detect CD62L (clone MEL-14), CD44 (clone IM7), CD25 (clone PC61.5), GATA-3 (TWAJ) and Thy1.1 (clone HIS51) (all from eBioscience or BD). IL-7Rα was detected by staining with anti-CD127-biotin (clone A7R34, eBioscience) and streptavidin-PE or -APC (eBioscience). Fluorescence intensities of stained cells were measured with a FACScalibur or LSRII flow cytometer and data analyzed with CellQuest or FlowJo software. For intracellular cytokine staining, splenocytes were restimulated ex vivo for 4 h with 1 μg/ml OVA peptide in the presence of 10 μg/ml BrefeldinA (Epicentre Biotechnologies) for the last 3 h and stained with PE-conjugated anti-IL-2 (clone JES6-SH4), -IL-4 (clone 11B11) and -IFN-γ (clone XMG1.2) mAbs (eBioscience or BD Pharmingen), using a Cytofix/Cytoperm kit (BD) according to the manufacturer’s instructions. To follow cell division during priming, naïve CD4⁺ T cells were labeled with 1 μM CFSE (Molecular Probes) for 10 min at room temperature. On day 4 after adoptive transfer and priming, CFSE content of KJ1–26⁺CD4⁺ cells was determined by flow cytometry.