CLSM and flow cytometric analysis of anti–G-CSF-R antibody internalization in 32D.cl8.6 cells expressing wt or [d715]G-CSF-R in the absence or presence of G-CSF. (A-B) Cells expressing wt (A) or [d715]G-CSF-R (B) were incubated with anti–G-CSF-R antibodies for one hour at 37°C, in the absence (left panels) or presence (right panels) of G-CSF (100 ng/mL), spun down on glass slides, fixed, permeabilized, and incubated with TRITC-conjugated goat antimouse antibodies before examination by CLSM. Original magnification, × 600. (C-D) Flow cytometric analysis of internalized biotinylated anti–G-CSF-R antibodies in 32D cells expressing wt (C) or [d715]G-CSF-R (D) in the absence (left panels) or presence (right panels) of G-CSF (100 ng/mL). 32D cells were allowed to bind biotinylated anti–G-CSF-R antibodies for one hour at 4°C and were subsequently incubated at 37°C for different time periods before staining with SA-PE to determine surface-bound G-CSF-R. Bold line indicates 0 minutes; dotted line, 15 minutes; thin line, 30 minutes; dashed line, 60 minutes; and shaded histogram, 0 minutes, in the absence of anti–G-CSF-R antibodies.