SP thymocytes are poor TNF producers relative to naïve splenic T cells during TCR activation in vitro.
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Thymocytes and splenocytes from P14-CD8+, OT-1-CD8+, SMARTA-CD4+ and OT-2-CD4+ TCR transgenic mice were stimulated in vitro as indicated for 4 hours and then stained for intracellular TNF cytokine, as described in Materials and Methods. For analysis, the cells were gated on either SP CD8+ CD4− or SP CD4+ CD8− cells. A, The percentages of TCR transgenic thymocytes and splenic T cells (both CD44lo and CD44hi) staining positive for TNF are shown. B, Resting P14-CD8+ thymocytes and naïve splenocytes were stained with mAbs to Vα2 and Vβ8.1. The profile for the SP thymocytes is shown in gray solid histograms and for splenic T cells in black line histograms. C, P14-CD8+ thymocytes and naïve splenocytes were either unstimulated (gray solid histograms) or stimulated (black line histograms) for 4 hours with GP33+αCD28 in vitro and stained with mAbs to the indicated surface molecules. D, CD45.1+ SP P14-CD8+ thymocytes were enriched and stimulated with GP33+αCD28 for 4 hours either alone or in the presence of live or irradiated (3000cGy) H-2Db WT or H-2Db KO splenocytes respectively. Cells were then stained for intracellular TNF. The percentages of CD45.1+ CD8+ thymocytes (CD44lo and CD44hi) staining positive for TNF are shown.