Surface CD38 clusters at the T/APC contact region. (A) Raji cells were labeled with CMAC blue and incubated in the presence (or absence) of SEE (1 μg/mL). Raji and Jurkat D8 cells were mixed at a 1:1 ratio, fixed, and double-stained for CD38 (green) or CD3-ζ (red; top and middle panels), or double-stained for CD45 (green) or CD3-ζ (red; bottom panels). Cells were examined using a Leica DMR photomicroscope (Mannheim, Germany; 63×/1.32-0.6 NA oil objective). (B) Percentage of Jurkat D8–Raji cells conjugates with surface CD38 redistributed at the contact zone relative to the total number of conjugates in the absence (□), or in the presence (■) of SEE. Data from 1 of 3 independent experiments, analyzing more than 80 conjugates. (C) Jurkat D8–Raji cell conjugates were generated in the presence of SEE, double-stained for CD38 (green) and CD3-ζ (red), and analyzed by a Leica TCS-SP confocal scanning laser microscope (63×/1.32-0.6 NA oil objective). Shown are the stacked pictures of 23 sections with 0.4-μm thickness. (D) T/DC conjugates were stained and examined as in panel A. CD38 and CD3-ζ staining is also shown in an intensity color-coded image, and the histogram analyzes fluorescence intensity along the line depicted on the image. (E) Percentage of T/DC conjugates with surface CD38 or total CD3-ζ redistributed at the contact zone relative to the total number of T/DC conjugates in the absence (□) or presence (■) of SEB. A total of 150 conjugates of each category were analyzed. In all panels for double staining, intact cells were incubated with the primary mAb followed by an Alexa Fluor 488–goat anti-mouse IgG, saturated with mouse serum. CD3-ζ was detected by permeabilizing cells (0.5% Triton X-100) prior staining with the anti–CD3-ζ 488 rabbit antibody, followed by the incubation with a Rhodamine Red X–labeled goat anti-rabbit IgG (H + L) highly cross-adsorbed.