Figure 5.

SKBr3 cell surface fluorescence quenching facilitates ErbB2 antibody uptake measurements and imaging. (A) Cells were surface-labeled on ice with 488-trastuzumab, washed, and then detached on ice (no 37°C incubation) as described in Materials and Methods. Cells were then sedimented and resuspended with the indicated concentrations of anti-Alexa-488 IgG on ice. Mean fluorescence intensity was measured by flow cytometry as described in Materials and Methods. Results were normalized to that of the control (nonquenched) sample. (B) Cells were surface-labeled with 488-trastuzumab or 488-pertuzumab, washed, and incubated at 37°C for the indicated intervals to internalize the surface-bound fluorescence. Cells were then rapidly chilled, detached, sedimented, and surface-quenched on ice with 25 μg/ml anti-Alexa 488 IgG, and % internalization calculated from flow cytometric data as described in Materials and Methods. Inset: rapid phase (0–5 min) portion of the data. (C) Cells were incubated continuously for 120 min with soluble 488-trastuzumab, 488-pertuzumab, or their corresponding 488-Fab fragments and then processed as in B to calculate % internalized. (D) Cells with surface-bound 488-trastuzumab were pulsed at 37°C for 5 min, rapidly chilled, surface-quenched, PFA-fixed, and then processed for immunofluorescence microscopy to detect surface quenching antibody using 647-anti-rabbit IgG as described in Materials and Methods. For comparison, the left panel inset shows 488-trastuzumab after a 15-min pulse but no surface quenching (20-fold lower exposure). Scale bars, 20 μM.