Representative data illustrating the phenotype of thymocytes and classification of early and late ACs. Freshly isolated thymocytes were stained with CD11b, B220, CD4 and CD8α and analysed by flow cytometry to assess the phenotype of the cells used to generate ACs. Minimal staining for B220 (A) and CD11b (B) was found. Approximately 98% of cells gated to exclude debris are lymphocytes and either CD4+, CD8α+ or CD4+CD8α+(C). Prior to administration of ACs cell viability was assessed by Annexin-V and Propidium Iodide (PI) staining and flow cytometry. Overnight culture alone induced predominantly early ACs (47% Annexin-V+ PI-) (D) whilst the addition of 1 μM dexamethasone elicited a population of late ACs (64.7% Annexin-V+ PI+) (E). Data representative of all AC preparations.