Self-reactive T cells accumulate phosphorylated TCR and SLP-76 into contact areas with the lipid bilayer despite little or no visible pMHC accumulation. T cells were incubated on lipid bilayers with ICAM-1 and pMHC. Cells were fixed after 5 or 30 min and stained for pCD3ζ or pSLP-76. TIRF microscopy (TIRFM) was used to visualize pCD3ζ or pSLP-76. (A–D) pCD3ζ staining, pMHC fluorescence, and images for IRM are shown at 5 min (A) or 30 min (C). pCD3ζ fluorescence at cell contact areas (defined by IRM images) at 5 min (B) or 30 min (D) was quantified and normalized to the value of HA:D7 fluorescence at 5 min. For each clone, 51–127 individual cells were analyzed. (E) Specificity of microcluster detection. Controls included: staining with isotype control antibody, treatment with Src kinase inhibitor PP2, and stimulation with irrelevant CLIP-MHC. Representative images at 5 min are shown. (F and G) pSLP-76 staining and pMHC accumulation at 5 min after stimulation with pMHC or CLIP-MHC. pSLP-76 fluorescence was quantified (G), as described in B. Data are representative of at least two independent experiments. Error bars in B, D and G represent the standard error of mean. Bars, 5 µm.