CD38-GFP in recycling endosomes and in the Golgi is targeted to the T/APC contact zone. (A) CD38-GFP–transfected J77 cells were incubated with Raji B cells pulsed with medium alone (top) or with SEE (bottom). Cells were then fixed, permeabilized, and stained with anti-CD71 (TfR) mAb followed by a goat anti-mouse Rhodamine Red X–second antibody. Samples were analyzed by a Leica TCS-SP5 confocal scanning laser microscope. Single confocal sections were taken of cells using fluorescence in GFP and rhodamine channels. Bars represent 5 μm for all panels (63×/1.4 NA oil objective). The white arrowheads point to intracellular CD38-GFP. The arrows point to the plasma membrane CD38-GFP or CD71. (B) CD38-GFP–transfected J77 cells were treated as in panel A except that after fixation and permeabilization, cells were stained with an anti-Golgi mAb followed by a goat anti-mouse Rhodamine Red X–second antibody. Samples were analyzed by confocal microscopy as in panel A. The white arrowheads point to the intracellular CD38-GFP, or the position of the Golgi apparatus. The arrows point to the plasma membrane CD38-GFP accumulated at the synapse. The acquired CD38-GFP (green), Golgi (red), the merge images, close-ups of some merge images, and DIC images are shown. A semiquantitative assessment of the level of colocalization was done by analyzing the colocalization scatter plot and generating a mask of the area of interest (AOI; right panels).