The expression of Thy-1 antigen and its mRNA in thymic DCs, splenic DCs, and DCs generated from Lin⁻c-kit⁺ HPCs.Ia⁺CD11c⁺CD3⁻CDB220⁻Gr-1⁻NK1.1⁻TER-119⁻ thymic and splenic DCs were highly purified by sorting them from low-density thymocytes and splenocytes after centrifuging on 14.5% metrizamid medium. The expression of Thy-1 antigen was reanalyzed on either freshly isolated or cultured thymic and splenic DCs upon stimulation with GM-CSF + mouse TNFα for 3 days. The percentage of Thy-1⁺ cells is indicated in the histograms (A). Thy-1 mRNA was examined in the indicated cells using RT-PCR (B). Total RNAs were extracted from 1 × 10⁵ cells of splenocytes, thymocytes, splenic DCs, thymic DCs, and Lin⁻c-kit⁺ HPC-derived DCs. DCs generated from Lin⁻c-kit⁺ HPCs were further sorted into Thy-1⁺ and Thy-1⁻ subpopulations. The β-actin transcripts were used as control (B). Similar experiments were performed three times and representative results are shown here.