CD4-independent internalization of the CXCR4-gp120 complex from the cell surface of a CD4⁻/CXCR4⁺ cell line. To assess CXCR4 (A) and CD4 (B) receptor surface expression by single-color flow cytometry analysis, cells were incubated for 1 hour at 4°C with either medium alone (control cells; a), the anti-CXCR4 12G5 MoAb (A; b), or the anti-CD4 MoAb OKT4a (B; b). Cells were stained with an anti-IgG mouse FITC. To analyze the subcellular localization of CXCR4 and gp120, CHO-K1 cells were incubated with 10 μg/mL of gp120 wt (C) or medium alone (D) for 1 hour at 37°C. After fixation and permeabilization, cells were stained with a rabbit anti-gp120 antiserum and a Texas Red-conjugated antirabbit IgG as well as the FITC-conjugated anti-CXCR4 12G5 MoAb, such that the intracellular presence of gp120 was observed as a red fluorescence and that of CXCR4 as a green fluorescence. The superposition of the two fluorochromes (yellow) is indicative of a colocalization of gp120 and CXCR4. This colocalization can only be observed in cells in which endosomes are observed in the microscopic confocal laser section.