Supplementary MaterialsTransparent reporting form. NanoSIMS imaging studies revealed the plasma membraneCderived particles are enriched in accessible cholesterol (a mobile pool of cholesterol detectable with the altered cytolysin ALO-D4) but not in sphingolipid-sequestered cholesterol [a pool detectable with ostreolysin A (OlyA)]. The finding that macrophages launch cholesterol-rich particles during cellular locomotion is likely relevant to cholesterol efflux and could contribute to extracellular cholesterol deposition in atherosclerotic plaques. boxed region in the live-cell image and in the low-magnification SEM image is demonstrated in the SEM image within the much right. Three self-employed experiments were performed; representative images are shown. Level pub, 5 m. Number 1figure product 1. Open in a separate window Macrophages launch particles from your plasma membrane of filopodia and lamellipodia by a process that resembles budding.(A) Top remaining, scanning electron micrograph (SEM) of a mouse peritoneal macrophage (arrow), revealing a lawn CYT387 sulfate salt of?~30-nm particles about the surrounding substrate. A higher magnification image of the region in the package is demonstrated in the top right image, centered on macrophage filopodia. Higher magnification images of the areas in the and boxes are demonstrated in the lower two images. (B) Upper left, SEM CYT387 sulfate salt of a mouse peritoneal macrophage (arrow), revealing a lawn of?~30-nm particles about the surrounding substrate. A higher magnification image of the region in the package is demonstrated in the image within the top right, centered on the lamellipodium of the macrophage. Higher magnification images of the areas in the and boxes are demonstrated in the images below. For both panels, arrows display the formation and launch of particles from macrophage filopodia and lamellipodia. Three independent experiments were performed; representative images are shown. Level bar for the top two CYT387 sulfate salt images, 2 m. Level bar for the bottom two images, 500 nm. Number 1video 1. arrow in video clips point to the cell that was visualized by SEM; the arrow in the video clips points to a region of lamellipodia projection/retraction; the package depicts the region of the cell that was consequently visualized by scanning electron microscopy (SEM) (observe Number 1). Video?shows a macrophage that was imaged by SEM in the top row of Number 1. Video shows a 24-h period of live-cell imaging. Number 1video 2. sequestered by sphingolipids), we performed live-cell imaging of Natural 264.7 macrophages (Figure 3videos 1C2) and then incubated the Rabbit Polyclonal to NCAPG2 cells with [15N]ALO-D4. The macrophages were then processed for SEM and NanoSIMS imaging. The lawn of particles around macrophages, visible by SEM, was enriched in 15N, as exposed by NanoSIMS imaging (Number 3). Of notice, the degree of 15N enrichment was higher in the lawn of particles than within the plasma membrane covering the macrophage cell body or macrophage filopodia (Number 3). Open in a separate window Number 3. Correlative live-cell, scanning electron microscopy (SEM), and NanoSIMS imaging, exposing that particles released onto the substrate during movement of filopodia and lamellipodia are enriched in accessible cholesterol.RAW 264.7 macrophages were plated onto iridium- and poly-D-lysineCcoated gridded glass-bottom Petri dishes. Videos were recorded for 24 hr at 5 min intervals (observe Number 3videos 1C2). The Live cell images in this number show the final frame of the videos, with the arrows pointing to the cells that were consequently visualized by SEM and NanoSIMS. After live-cell imaging, cells were incubated with [15N]ALO-D4 (a altered cytolysin that binds to accessible cholesterol). The same cells that were imaged by live-cell imaging were consequently imaged by SEM (to visualize particles) and NanoSIMS (to visualize [15N]ALO-D4 binding). The particles left behind within the substrate during movement of lamellipodia and filopodia bound [15N]ALO-D4 avidly. 12C14NC NanoSIMS images were used to visualize cell morphology; the 15N/14N images show 15N enrichment (arrows point to the projection and retraction of the lamellipodia. box indicates the region that was consequently imaged by scanning electron microscopy (observe Number 3). Video?shows a macrophage imaged by SEM and NanoSIMS in the top row of Number 3. Video shows the final 12 h of a 24-h period of live-cell imaging. Number 3video 2. and CYT387 sulfate salt boxes are demonstrated on the right. Composite images (box is demonstrated on the right. arrows point to platinum nanoparticles binding to macrophage particles. Three independent experiments were?performed; representative images are shown. Level pub, 1 m. Number 4figure product 1. Open in a separate window Particles released from non-biotinylated macrophages do not bind streptavidin-conjugated platinum nanoparticles as judged by scanning electron microscopy (SEM).Non-biotinylated macrophages were plated onto glass-bottom Petri dishes. On the following day time, the cells were incubated with streptavidin-conjugated 40-nm platinum nanoparticles. Cells were then fixed with 1% glutaraldehyde and processed for SEM. Secondary electron (SE).