Figure 10. Biotinylated mouse peritoneal macrophages release plasma membrane–derived material onto the surface of dead endothelial cells.

Mouse brain endothelial cells (bEnd.3) were plated onto glass- bottom Petri dishes and allowed to grow to confluency. After fixing the endothelial cells with 0.1% glutaraldehyde in PBS, they were washed extensively with PBS. Next, biotinylated macrophages (i.e., macrophages in which the cell-surface proteins had been biotinylated with Sulfo-NHS-SS-biotin) were plated onto bEnd.3 cells. Secondary electron (SE) and backscattered electron (BSE) images were obtained with a scanning electron microscope. As expected, the SEM images revealed binding of the streptavidin- conjugated gold nanoparticles to both the cell body and filopodia of macrophages. In addition, gold nanoparticles were observed on the surface of adjacent endothelial cells. BSE images were helpful in identifying gold nanoparticles. Higher magnification images of the blue and red boxed regions in each image are shown below. As an experimental control, bEnd.3 cells without macrophages were fixed and incubated with streptavidin-conjugated gold nanoparticles. Higher magnification images of the red boxed regions are shown on the right. No binding of gold nanoparticles was detected. Three independent experiments were performed; representative images are shown. Scale bar, 2 µm.

Figure 10—figure supplement 1. Correlative live-cell and SEM images, revealing the release of particles by a live macrophage onto the surface of a dead macrophage.

RAW 264.7 macrophages were plated onto poly-D-lysine–coated gridded glass-bottom Petri dishes, and videos of cell movement were recorded for 20 hr at 5 min intervals (see Figure 10—video 1). Live-cell microscopy revealed a live macrophage extending its lamellipodium (red arrow in the video) over both the surrounding substrate and over the dead macrophage (white arrow in the video). The white box in the video shows the region that was imaged by SEM. The SEM revealed that vesicular particles had been released onto both the surrounding substrate and onto the surface of the dead macrophage. Scale bar for the SEM on the top right, 5 μm. The region encompassed by a red box in the SEM on the top right is shown at higher magnification in the SEM on the lower left. Scale bar for the lower left SEM, 5 μm. The red boxed areas in the lower left and lower middle SEMs are shown at higher magnification on the right. Images shown are from one experiment. Scale for lower middle and lower right images, 500 nm.

Figure 10—video 1. Release of vesicular particles by a live RAW 264.7 macrophage onto the surrounding substrate and onto a dead RAW 264.7 macrophage.

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DOI: 10.7554/eLife.50231.048

RAW macrophages were plated onto poly-D-lysine–coated glass-bottom Petri dishes and incubated in media containing 10% FBS. Cells were imaged by live-cell microscopy for 20 hr at 5 min intervals. White arrow points to a dead macrophage; red arrow points to the projection and retraction of the live macrophage lamellipodia over the dead macrophage; white box indicates the region that was subsequently imaged by SEM (see Figure 10—figure supplement 1). Video shows a 20 hr period of live-cell imaging.