Figure 2.
Figure 2. Analysis of neutrophil abluminal crawling in vivo. Cremaster muscles of αSMA-RFPcherry x Lys-EGFP-ki mice were stimulated with TNF for 2 h before being exteriorized and visualized by live confocal microscopy. (A) Time-lapse images showing a neutrophil (green) crawling along pericyte processes (red) from its site of TEM (i.e., starting point of the time course analysis, blue arrow) toward a gap between adjacent pericytes (red arrow). In the top panels, the neutrophil and pericyte layer are viewed from outside the vessel. The crawling path is shown with the yellow line. An isosurface mask was created to enable clearer visualization of the migrating neutrophil within the pericyte layer. In the bottom panels, 2-µm cross-sections in the z-axis show the position of the migrating neutrophil relative to the EC layer (blue) in the first image showing that it has passed the endothelium. The subsequent images show the neutrophil in relation to pericytes (red) during its abluminal crawling as captured at multiple time points. (B) Time-lapse images showing another example of neutrophil crawling along pericyte processes and avoiding the gaps between adjacent pericytes. Crawling path (yellow line) and directionality (black arrow) are shown in the top panels; the vessel segment is viewed in 3D from outside the vessel. The bottom panels show a higher magnification maximal intensity projection (2D) of the vessels with the shape of the neutrophil drawn with a green dotted line at each of the time points to illustrate its shape and its position relative to pericyte processes. (C) Four examples of postcapillary venules (pericyte in red) viewed in 3D from the extravascular space (top) or at a z-section angle (bottom), together with individual crawling paths (green line) and directionality (black arrow) of the neutrophil being tracked along pericyte processes in 3D. For clarity, the neutrophil being tracked is not depicted. Examples 1 and 2 show both the migration path and directionality of cells being tracked in A and B, respectively. All corresponding track and displacement lengths are given below the images. Bars, 10 µm.

Analysis of neutrophil abluminal crawling in vivo. Cremaster muscles of αSMA-RFPcherry x Lys-EGFP-ki mice were stimulated with TNF for 2 h before being exteriorized and visualized by live confocal microscopy. (A) Time-lapse images showing a neutrophil (green) crawling along pericyte processes (red) from its site of TEM (i.e., starting point of the time course analysis, blue arrow) toward a gap between adjacent pericytes (red arrow). In the top panels, the neutrophil and pericyte layer are viewed from outside the vessel. The crawling path is shown with the yellow line. An isosurface mask was created to enable clearer visualization of the migrating neutrophil within the pericyte layer. In the bottom panels, 2-µm cross-sections in the z-axis show the position of the migrating neutrophil relative to the EC layer (blue) in the first image showing that it has passed the endothelium. The subsequent images show the neutrophil in relation to pericytes (red) during its abluminal crawling as captured at multiple time points. (B) Time-lapse images showing another example of neutrophil crawling along pericyte processes and avoiding the gaps between adjacent pericytes. Crawling path (yellow line) and directionality (black arrow) are shown in the top panels; the vessel segment is viewed in 3D from outside the vessel. The bottom panels show a higher magnification maximal intensity projection (2D) of the vessels with the shape of the neutrophil drawn with a green dotted line at each of the time points to illustrate its shape and its position relative to pericyte processes. (C) Four examples of postcapillary venules (pericyte in red) viewed in 3D from the extravascular space (top) or at a z-section angle (bottom), together with individual crawling paths (green line) and directionality (black arrow) of the neutrophil being tracked along pericyte processes in 3D. For clarity, the neutrophil being tracked is not depicted. Examples 1 and 2 show both the migration path and directionality of cells being tracked in A and B, respectively. All corresponding track and displacement lengths are given below the images. Bars, 10 µm.

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