TCR and CD28 stimulation induced ROS production and increased T cell apoptosis. CD4+CD25- T cells were stimulated with anti-CD3 and anti-CD28 antibodies for the indicated time points and then intracellular ROS was stained with DHE. The mean fluorescence intensity (MFI) of DHE in a single cell was measured with FACS. A aliquot of cells was stained with Annexin-V and 7-AAD to analyze the early apoptotic (Annexin-V+7-AAD-) and late apoptotic/dead (Annexin+7-AAD+) cells. The cells from each cultured well were also examined for viable cells by trypan blue exclusion assay. A. A representative histogram profile of DHE staining on the different days. The filled histogram is the un-labeled cells (negative control). B. The values are displayed as the Mean ± SD of the MFI of DHE between stimulated (αCD3+αCD28) and non-stimulated (medium) live T cells (R1 gated cells in Fig. S1) at the indicated time points (n = 2 to 5). C. The values are presented as the Mean ± SD of the early apoptotic (Annexin+7-AAD-) and dead/late apoptotic (Annexin+7-AAD+) between anti-CD3 and anti-CD28 (3+28) and non-stimulated (medium) cells (n = 2 to 5). D. The values are shown as the Mean ± SD of the live cells (trypan blue negative) per well. The original cell number was 1 × 106 per well(24-well plate; n = 2 to 5).