TCR–CD3 engagement induces open-CD3 in B6 thymocytes. B6 thymocytes were treated in vitro with either Ham IgG (basal condition) or anti-CD3ε mAb 2C11 (stimulated condition). Next, cells were lysed and either total lysate (TL) or CD3-PD assay samples were prepared (see Materials and Methods), involving SDS-PAGE and WB for CD3ζ. Twenty-two independent CD3-PD assays were performed, and open-CD3 induction was calculated in each as the ratio of open-CD3 found in stimulated relative to basal conditions. A–C, Three independent experiments are shown representing low (A), intermediate (B), and high (C) levels of induced open-CD3 by 2C11 stimulation. D, Values of open-CD3 induction achieved by 2C11 stimulation are plotted (mean = 4.7, median = 4.4, SD = 2.6, SE = 0.5).