Content mixing of HIV-1 Env-expressing HeLa cells and CD4-X4-expressing NIH3T3 cells in the absence (A) or presence (B) of gp41e. For content mixing, HIV-1 Env-expressing HeLa (effector) cells were incubated with CD4-X4-expressing NIH3T3 (target) cells. CMTMR-labeled target cells (red) were co-cultured with calcein-labeled effector cells (green) at 37°C, and dye redistribution was monitored microscopically as described in the Materials and Methods. Content mixing occurred at about 20 minutes after CD4-X4 target cells binding in the absence of gp41e (A, arrow), but not in the presence of gp41e (B). Note that the lower two arrows in panel (A) clearly indicate content mixing while the upper three likely result from overlapping cell images. Scale bar is 10 μm.