Figure 3.

Changes in intracellular calcium in DP thymocytes and T cells stimulated with various ligands. (A) Dot plots of FL4/FL5 versus time for preselection (OT-I TAP^o) DP thymocytes stimulated with APC alone, anti-CD3 mAb + goat anti–mouse Ig, OVAp + APC, and G4 + APC. The break in the trace corresponds to the time where the T cells and APCs were centrifuged together before being resuspended gently to pass through the flow cytometer. The two breaks for mAb cross-linking correspond to the sequential addition of anti-CD3 mAb and the goat anti–mouse Ig. (B) Representative histograms for FL4/FL5 of preselection DP thymocytes with APC alone or 10 μM OVAp, showing nonoverlapping markers set for low, intermediate, high, and very high expressing cells corresponding to increased intracellular calcium levels. Histogram data are based on the gate shown in A. (C) Graphs of the percentage of cells in each FL4/FL5 gate for preselection DP thymocytes and OT-I CD8 SP splenocytes stimulated with anti-CD3 mAb, OVAp, and G4 peptide. Markers were set as in B. Background levels determined by the APCs alone control have been subtracted from the values graphed. The APCs used in this experiment were of the 5AK^b cell line. Similar results were seen using TAP^o PEC as the APCs. The data shown are representative of four independent experiments.