Thymocyte and Splenic Cell Immunophenotyping

TECs were isolated by flow cytometry using the protocol of Gray et al [10 (link)]. Single cell suspensions of thymocytes and splenic cells were stained with the fluorochrome-conjugated antibodies as described [11 (link)]. For intracellular staining, the cells were first permeabilized with a BD Cytofix/Cytoperm solution for 20 minutes at 4°C. Direct or indirect staining of fluorochrome-conjugated antibodies included: CD4, CD8, CD25, CD44, CD62L, c-kit, IL-7Rα, BP-1, CD45, I-A, H-2K^b, CD45.1, Ki67, CD69, EpCAM1, IL-2, IFN-γ, TNFα, Bcl-2, and a panel of TCR Vβ clonotypes (BioLegend, BD Biosciences, San Jose, CA, or eBioscience, San Diego, CA), as well as Bcl-xL (Cell Signaling Technology, Inc., Danvers, MA). ETPs were identified by phenotypic analysis (Lineage^- c-kit⁺ IL-7Rα^- CD44⁺CD25^-) as described [8 (link),11 (link)]. An antibody cocktail, composed of antibodies against TER-119, B220, CD19, IgM, Gr-1, CD11b, CD11c, NK1.1, TCRβ, CD3e, and CD8α, was used to identify lineage negative cells. Annexin V and terminal deoxynucleotidyltrasferase dUTP nick end labeling (TUNEL, APO-DIRECT) apoptosis detections kits were purchased from BD Biosciences. The samples were analyzed on a FACSCalibur or LSR II flow cytometer (BD Biosciences).