Figure 3
The localization of intracellular CD63 and F-actin in stimulated neutrophils treated in the presence of R59022. Primed neutrophils (2.5 × 105) were treated with ANCA IgG (200 µg/mL) for 15 min. Paired samples of cells were also treated with R59022 (18 µmol/L) for 10 min before priming. Cells were fixed and labeled with the plasma membrane stain wheat-germ agglutinin (alexa-fluor 594 conjugated) and then permeabilized and labeled with anti-CD63 (FITC conjugated) or phalloidin (FITC conjugated), spun onto microscope slides and visualized by confocal microscopy at 100× magnification, with an additional 3.5x digital zoom. One representative cell is shown for each treatment (with over 75% of cells exhibiting this phenotype) in blocks of three, comprising FITC labeling in green, plasma membrane labeling in red and merged images with areas of colocalization in yellow on the image. All merged images were analyzed for distribution of fluorescence intensity of anti-CD63-FITC or phalloidin-FITC staining with the plasma membrane wheat-germ agglutinin alexa-fluor 594 conjugate stain within the same cell. An arrow was placed (cross-sectional) over the cell and histograms generated to distinguish fluorescent intensities of both FITC and alexa-fluor staining along the line of the arrow. Red histograms represent alexa-fluor membrane staining, whereas the green histograms represent FITC staining. The y axis represents fluorescence intensity, whereas the xaxis represents distance across the cell (µm). All neutrophils in the image are from the same donor and the same individual experiment. The figure is representative of three individual experiments.