Restricted area of the cSMAC defined by the APA1/1 epitope. (A) Localization of APA1/1 staining in the cSMAC. Jurkat T cells were incubated for 15 minutes with SEE-treated Raji APCs. The APCs were prestained with CMAC (blue), and the T-cell/APC conjugates were stained with the markers indicated in green (top row) and red (middle row). Cell conjugates were examined with a charge-coupled device (CCD) camera at a magnification of 100 ×. P-Tyr indicates antiphosphotyrosine antibody 4G10. (B) A differential interference contrast (DIC) image of the T cell/APC conjugate is shown to illustrate the position of the IS. APA1/1 marks a restricted area within the cSMAC. Jurkat-Raji cell conjugates were prepared as above and triple stained for APA1/1 (green channel), CD3ϵ cytoplasmic tail M20 (red channel), and CD3ζ (far red channel). Each channel was analyzed separately to provide a color-coded scale of intensities (red represents the strongest signal). (insets) 1.5-fold magnifications of the IS. Images were acquired with a Zeiss Axiovert 200 microscope.