Fig. 1. Measuring ErbB2 internalization in single cells and cell populations in a non-invasive manner. (A) The method used to study ErbB2 internalization and cleavage microscopically. The upper image shows fixed, non-permeabilized cells that are stained with an antibody (Sc08) recognizing an extracellular ErbB2 epitope followed by Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (GM-488) (green, this staining is termed ErbB2_surface). The lower image shows cells that subsequently are permeabilized and stained with Sc08 again plus Alexa Fluor 568-conjugated GM secondary antibody (GM-568) (red, this staining is termed ErbB2_total) and an antibody recognizing a cytoplasmic C-terminal ErbB2 epitope (2242) followed by Alexa Fluor 633-conjugated goat-anti-rabbit secondary antibody (GR-633) (blue, this staining is termed ErbB2_Cterminal). (B,C) Confocal images of SK-BR-3 cells stained for ErbB2 as described in A. ErbB2 is internalized after antibody crosslinking of ErbB2 (B). This was done by incubating the cells with Sc08 for 5 minutes followed by GM for 30 minutes at 37°C. Similarly, ErbB2 is internalized after 3 µM geldanamycin treatment as indicated (C). Arrows in the enlarged image indicate that surface exposed ErbB2 tends to aggregate before internalization. (D) Scatter diagrams of pixel intensities from an untreated cell and a cell treated with 3 µM geldanamycin for 2 hours and stained as in A. The x-axis depicts the fluorescence intensity of ErbB2_surface (green), whereas the y-axis depicts the fluorescence intensity from ErbB2_total (red). The analyzed cells are shown in the upper right corners of the scatter diagrams, and the region of interest is encircled in a green oval. The blue overlay represents pixel intensities that are below the threshold and therefore excluded from the analysis. The Pearson's correlation coefficients, r²_i, are calculated from all pixels above the threshold in the selected area and shown in the top of the diagram. Thus, reduced r²_i reflects internalization because of less correlation between ErbB2_surface and ErbB2_total. (E) Histograms of the colocalization values for ErbB2_surface and ErbB2_total (r²_i) from SK-BR-3 cells treated with 3 µM geldanamycin as indicated and stained as described in A. Note that increased amounts of internalized ErbB2 are seen with time after geldanamycin treatment (decreasing r²_i values). (F) Histogram of the r²_i values from SK-BR-3 cells where ErbB2 is internalized after antibody crosslinking as in B. Cells were stained as described in A. Bars, 20 µm.