A pool of internalized IL-7Rα recycles to the cell surface. (A) Colocalization between IL-7Rα and Rab-11 recycling endosomes was assessed after 5 minutes of stimulation with IL-7 (50 ng/mL). Total IL-7Rα expression was assessed by intracellular staining as for flow cytometry. Rab-11–positive vesicles were further detected by incubation with rabbit α-human Rab-11 unconjugated antibody followed by secondary detection with α-rabbit IgG Alexa 488. Coverslips were mounted over Vectashield/DAPI. Cells representative of 3 independent experiments are shown. (B) Colocalization between IL-7Rα and endosomes was performed by antibody chase and colocalization assay, as described in “Antibody ‘chase’ for assessment of receptor colocalization.” Briefly, all cells, plated onto coverslips, were initially incubated at 4°C with unconjugated α-human IL-7Rα antibody for 30 minutes and subsequently washed and transferred to 37°C in the presence or absence of IL-7 (50 ng/mL) for 1 hour to allow for IL-7Rα internalization. Cells were washed, and the positive control (time 0) was generated by incubating the cells at 4°C with α-mouse IgG Alexa 488 secondary antibody for 30 minutes. The remaining coverslips underwent an acid wash treatment to remove surface-bound antibody. The negative control was not further processed. The primary antibody/receptor complex was allowed to recycle to the cell surface in the experimental conditions, by switching the cells to 37°C again for 1 hour. Recycled IL-7Rα/antibody complex was detected by incubating the cells with anti–mouse IgG Alexa 488 secondary antibody for 30 minutes on ice. Cells were fixed, and the different coverslips mounted with Vectashield/DAPI. Representative images of 2 independent experiments are shown for each condition. (C) Microscopy images of the antibody feeding and recycling assays were quantified by determining the mean intensity of fluorescence (MIF) of at least 8 independent fields of view for each condition. **P < .01, ***P < .001 (Student t test, 2-tailed).