Flow cytometry of IFN-γR2 internalization in malignant T cells. (A) ST4 T cells cultured for 24 hours in the presence or absence of serum, or absence of serum in the presence of IGF-1 (100 ng/mL) were recovered and incubated with biotin-conjugated anti–IFN-γR2 mAb at 4°C(□)or 37°C(▪). After 4 hours, cells were permeabilized and stained with PE-conjugated streptavidin. (B) ST4 T cells, cultured for 24 hours in medium without serum in the absence or in the presence of scalar doses of IGF-1 (from 1 to 100 ng/mL), were incubated with unconjugated anti–IFN-γR2 mAb or with isotype-matched mAb at 37°C. After 4 hours, cells were permeabilized and stained with FITC-conjugated rabbit F(ab′)2 anti–mouse Ig. Mean specific internalized fluorescence was calculated by subtracting the mean internalized fluorescence obtained with specific anti–IFN-γR2 mAb from that obtained with isotype-matched mAb.
