Thymic Epithelial Cell Immunophenotyping

Thymic epithelial cells were acquired as described above. Cells were incubated with 2.4G2 before staining with other antibodies. The antibodies used included anti-CD45 (30-F11, eBioscience), anti-EpCAM (G8.8, Biolegend), anti-Ly51 (6C3, Biolegend), FITC labelled Ulex europaeus agglutinin-1 (UEA-1; Vector Laboratory), anti-IA/IE (M5/114.15.2, Biolegend), anti-SSEA-1 (MC-480, Biolegend), anti-LTβR (eBio3C8, eBioscience). C-CPE (C. perfringens enterotoxin) was produced as described47 (link) and biotin-conjugated; Streptavidin APC-eFluor 780 (eBioscience) was used for visualization of C-CPE. For intracellular staining of Ki-67 (B56, BD) and active caspase 3 (C92-605, BD), cells were fixed and permeabilzated with BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (554714) and stained according to the manufacture’s protocols. Fixable viability dye (L-34967, ThermoFisher) was used to exclude dead cells. For thymocyte and splenocyte analysis, cells were stained with anti-CD4 (RM4-5, eBioscience), anti-CD8 (53-6.7, eBioscience), anti-Va2 (B20.1, eBioscience), anti-Vb5 (MR9-4, eBioscience), and anti-CD24 (M1/69, Biolegend) before flow cytometry analysis. The samples were analyzed on BD LSRFortessa and FlowJo software (Tree Star Inc).