Fig. 4.

Phagotubules facilitate content transfer between LPS-containing phagosomes. WT BMDCs pulsed with a 1:1 mixture of LPS/OVA-TxR or LPS/OVA-647 beads and LPS/OVA-AF488 beads were chased as indicated. (A) DCs chased for 2.5 h were analyzed by live cell imaging. (Left) DIC (Upper) and corresponding immunofluorescence image (Lower) from a frame of a representative movie. (Right) Magnification of the boxed region from a frame sequence of the same movie. Arrowheads indicate green phagotubules that contact red phagosomes (arrows). (B) DCs were chased as indicated, after which intact phagosomes were isolated and analyzed by flow cytometry. (Left) Forward scatter (FSC) and side scatter (SSC) plots showing a gated region (box) representing single phagosomes. (Center) Dot plots of a gated region from each chase time point in a representative of three experiments. The percentage of bicolored phagosomes (upper right quadrant) is indicated in red. (Right) Mixing control. Phagosomes were isolated from DCs pulsed with either LPS/OVA-AF488 beads or LPS/OVA-AF647 beads alone, chased for 4 h, and then mixed before homogenization. (C and D) Untreated Myd88^−/−, Tlr4^−/−, or WT DCs or WT DCs treated with vehicle or inhibitors as indicated were chased for 4 h, and isolated phagosomes were analyzed by flow cytometry as in B. (C) The percentage of phagosomes bearing both AF488 and AF647 labels was quantified (mean ± SD) in five independent experiments. (D) Representative experiment.