Actin-perturbing drugs alter the binding of IgG-opsonized particles. (A) Representative differential interference contrast (DIC, top panels) and spinning-disk confocal fluorescence images (bottom panels) of RAW cells stably expressing actin-GFP. Where indicated the cells were treated with 1 µM jasplakinolide, 0.2 µM latrunculin, and/or 60 µM blebbistatin. DMSO was used as vehicle. Bars: 8 µm (top panels), 17 µm (bottom panels). (B) Quantitation of the effect of actin-perturbing drugs on particle binding. Data are presented as a binding index (see Materials and methods) normalized to the vehicle control and are means ± SEM of three experiments, each counting at least 150 macrophages per condition. (C and D) Surface expression of FcγRII/III (C) and FcγRI (D), measured by flow cytometry after pretreatment with actin-perturbing agents or vehicle only. (E) Binding of soluble IgG aggregates to RAW macrophages at 15°C. Representative xy and xz optical slices obtained by spinning-disk microscopy are illustrated; bar, 17 µm. Surface-associated IgG aggregates were detected using a Cy3-conjugated secondary antibody. (F) Quantification of surface-associated IgG in cells treated with actin-perturbing agents. Following labeling as in E, the cells were detached from the substratum, fixed, and analyzed by flow cytometry. Histograms in C–E are representative of two independent experiments of each type.