Fusion of azurophilic granules with phagosomes containing <i>S. pyogenes</i> bacteria.

Abstract

<p>A. Western blot of isolated phagosomes and cell lysate. Differentiated HL-60 cells were allowed to phagocytose IgG-opsonized, heat-killed and magnetically labeled <i>S. pyogenes</i> bacteria for 20 min at a bacteria/cell ratio of 5∶1. Following washes, the cells were lysed by nitrogen cavitation and phagosomes were retrieved by magnetic selection. Equal amounts of phagosomes were loaded and probed with antibodies against cathepsin D (azurophilic granule content marker) and GM130 (Golgi marker) with anti-<i>S. pyogenes</i> as loading control. Increasing amounts of cell lysate (relative protein content.05×, 0.5× and 1.0×) was used as a control. B. Azurophilic granule–phagosome fusion. Differentiated HL-60 cells were allowed to phagocytose opsonized Oregon Green-labeled <i>S. pyogenes</i> bacteria, either the BMJ71 (i-iii) or the AP1 (iv–v) strains, at a bacteria/cell ratio of 10∶1. After a synchronized presentation, the samples were incubated at 37°C for 5 min, fixed and incubated with antibodies directed against CD63, subsequently detected using Alexa 594 F(ab')₂ fragments. Single deconvolved focal planes from serial z-stacks were taken from the mid-part of the HL-60 cells. Oregon Green staining shows the localization of bacteria (ii, v). Magenta staining shows the localization of the azurophilic granule membrane marker CD63 (i, iv). The images are also presented as merged (iii, vi). Size bar: 5 µm.</p