Dynamic redistribution of ALCAM and CD6 during the formation of T-cell–APC conjugates. (A) Jurkat-CD6*RFP cells were mixed with CFSE-labeled K562 or K562-ALCAM cells in a 1:1 ratio and incubated for 20 minutes at 37°C. Cells were mounted on poly(l)lysine coated slides and analyzed by fluorescence microscopy. Scale bar represents 10 μm. (B-C) Jurkat-CD6*RFP cells were allowed to adhere onto fibronectin-coated slides and K562-ALCAM*GFP cells were added in a 1:2 ratio. Each time point shows the DIC images merged with the fluorescence images of the Jurkat and K562 cells taken from the video at the indicated time points. Time frames were selected based on clear redistribution events. Representative examples of ALCAM and CD6 relocalization at the Jurkat/K562-ALCAM interface (B) and the K562-ALCAM/K562-ALCAM interface (C) are shown. (D-E) Fluorescence intensity of ALCAM*GFP (♦) and CD6*RFP (▪) in a fixed region of the synapse between Jurkat and K562-ALCAM (D) or 2 K562-ALCAM cells (E). The fluorescence intensity in the synapse region (light blue) was divided by the average intensity measured in the whole cell (ALCAM-expressing K562 cells represented in green and CD6-expressing Jurkat cells in red), as indicated in the top panels. These values, representing the recruitment index (RI), were calculated for each imaging time point and plotted on the diagrams. RI values of 1 representative conjugate (out of 3 independent experiments) are shown.