Elevated TCR-induced Ca²⁺ response of FU-Lck transgenic thymocytes. Thymocytes were labeled with 2μM indo-1, and then stained with biotin-conjugated anti-TCRβ ± CD4 (RM4.5) Abs. Additional fluorescently conjugated CD8 and CD4 (clone YTA3.1 does not compete with clone RM4.5) Abs were used to identify thymocyte populations, and Ca²⁺ fluxes were monitored by flow cytometry. Baseline levels were determined for 60 seconds, and TCR ± CD4 cross-linking was achieved by the addition of streptavidin-allophycocyanin conjugates. Arrows on histograms indicate time of addition of streptavidin. Ca²⁺ traces in gated DP populations after cross-linking of TCR + CD4 are represented in the histograms shown in panel A, while those obtained after cross-linking of TCR alone are represented in panel B. In all cases, data represent 1 of 3 repeated experiments.