Cell-associated LPS.

<p><i>Aoah^+/+</i> or <i>Aoah^−/−</i> mice were given 10 µg LPS-FITC i.p. Ten days later, their peritoneal cells were harvested, fixed, and either permeabilized (<b>B</b>) or not (<b>A</b>). Anti-FITC Ab conjugated with Phycoerythrin (PE) was added to measure macrophage (F4/80+) surface (<b>A</b>) or total (<b>B</b>) LPS-FITC. Dotted lines, F4/80+ cells from mice that received LPS i.p. (control for autofluorescence); solid lines, F4/80+ cells from mice that were given i.p. LPS-FITC. Red lines, <i>Aoah^−/−</i> ; blue lines, <i>Aoah^+/+</i>. (<b>C</b>) The ratio of the GeoMFI of cell surface or total LPS-FITC to the GeoMFI of control macrophages. The dotted line indicates a ratio of 1.0 (no LPS-FITC association with cells). For both <i>Aoah^−/−</i> and <i>Aoah^+/+</i> cells, most of the FITC was intracellular. (<b>D–I</b>) Mice were given 10 µg LPS-FITC i.p. Ten days after injection, peritoneal cells were harvested and cultured in plates with coverslips for 18 hours. Floating cells were washed away and adherent macrophages were fixed, permeabilized, and stained with anti-FITC Alexa fluor 488 (green), anti-LAMP1 (CD107) Ab (red) and DAPI (blue). (<b>D and E</b>) <i>Aoah^−/−</i> macrophages. <b>F</b>, overlay of <b>D</b> and <b>E</b>. (<b>G and H</b>) <i>Aoah^+/+</i> macrophages. <b>I</b>, overlay of <b>G</b> and <b>H</b>. Much of the FITC-LPS was localized to LAMP1-positive vesicles in both <i>Aoah^−/−</i> and <i>Aoah^+/+</i> macrophages. Scale bar = 5 µm.