Figure 5
Figure 5. Directed mechanotactic crawling was compromised in HPK1-deficient PMNs. Mechanotactic crawling of isolated murine PMNs evaluated in vitro. (A-C) Flow chambers coated with P-selectin (10 µg/mL), ICAM-1 (12.5 µg/mL), and CXCL1 (5 µg/mL) were used. Flow of 1 dyne/cm2 was applied and crawling was recorded for 10 minutes by using live-cell imaging, followed by offline analysis with ImageJ. At least 635 cells were analyzed for each strain. n = 9 flow chambers with cells from 5 mice each. Diagrams show mean ± SD; *P < .05; n.s. = not significant. (D-G) Intraluminal leukocyte crawling in inflamed postcapillary venules, assessed by intravital microscopy of the mouse cremaster muscle 2.5 hours after intrascrotal injection of TNF-α (500 ng). At least 189 cells were analyzed for each strain; n = 6 venules from 4 mice each. Diagrams show mean ± SEM; *P < .05; n.s. = not significant. (A,D) Percentage of crawling cells compared with number of totally adherent cells. Cells that exceeded a displacement of more than one cell diameter (Euclidean distance) were considered as crawling. (B,E) Mean crawling velocities were analyzed offline in time-lapse videos with temporal resolution of 3 seconds (in vivo) or 5 seconds (in vitro). (C,F) Percentage of cells ending up in a 60° angle that is symmetrically divided by the vector of flow. (G) Representative microscopic images (upper panel) with single-cell migration tracks (colored arrows) of leukocytes crawling on the inflamed vessel wall of postcapillary venules in TNF-α–treated cremaster muscles of HPK1+/+ and HPK1−/− mice. Dotted white arrows indicate direction of blood flow and dotted green lines depict the vessel walls; bar = 50 µm. Rose diagrams (lower panel) were generated by overlaying the single-cell tracks of all tracked cells, rotating an angular sector with an interior angle of 90° by 1° steps, and counting the cells that ended up in that angle. The counts were grouped in 10° intervals with the radius of each wedge representing the accumulated cell number. The arrow indicates the direction of blood flow.

Directed mechanotactic crawling was compromised in HPK1-deficient PMNs. Mechanotactic crawling of isolated murine PMNs evaluated in vitro. (A-C) Flow chambers coated with P-selectin (10 µg/mL), ICAM-1 (12.5 µg/mL), and CXCL1 (5 µg/mL) were used. Flow of 1 dyne/cm2 was applied and crawling was recorded for 10 minutes by using live-cell imaging, followed by offline analysis with ImageJ. At least 635 cells were analyzed for each strain. n = 9 flow chambers with cells from 5 mice each. Diagrams show mean ± SD; *P < .05; n.s. = not significant. (D-G) Intraluminal leukocyte crawling in inflamed postcapillary venules, assessed by intravital microscopy of the mouse cremaster muscle 2.5 hours after intrascrotal injection of TNF-α (500 ng). At least 189 cells were analyzed for each strain; n = 6 venules from 4 mice each. Diagrams show mean ± SEM; *P < .05; n.s. = not significant. (A,D) Percentage of crawling cells compared with number of totally adherent cells. Cells that exceeded a displacement of more than one cell diameter (Euclidean distance) were considered as crawling. (B,E) Mean crawling velocities were analyzed offline in time-lapse videos with temporal resolution of 3 seconds (in vivo) or 5 seconds (in vitro). (C,F) Percentage of cells ending up in a 60° angle that is symmetrically divided by the vector of flow. (G) Representative microscopic images (upper panel) with single-cell migration tracks (colored arrows) of leukocytes crawling on the inflamed vessel wall of postcapillary venules in TNF-α–treated cremaster muscles of HPK1+/+ and HPK1−/− mice. Dotted white arrows indicate direction of blood flow and dotted green lines depict the vessel walls; bar = 50 µm. Rose diagrams (lower panel) were generated by overlaying the single-cell tracks of all tracked cells, rotating an angular sector with an interior angle of 90° by 1° steps, and counting the cells that ended up in that angle. The counts were grouped in 10° intervals with the radius of each wedge representing the accumulated cell number. The arrow indicates the direction of blood flow.

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