Skap2 is required for Mac-1–dependent intravascular crawling, extravasation, and chemotaxis of neutrophils. (A–D) Intravascular crawling of Ly-6G–labeled neutrophils in postcapillary venules of WT and Skap2^−/− mice during superfusion with CXCL2. (A) Percentage of adherent cells that crawled (A), crawling velocity (B), crawled distance (C), and representative images (D) are shown. The arrow indicates direction of movement. n = 4 mice/group. (E–G) Crawling of CXCL2-stimulated WT and Skap2^−/− neutrophils on serum-coated parallel plate flow chambers in vitro. Crawling velocity (E) and accumulated (F) and Euclidian (G) crawled distance before (preflow), during (flow), and after (postflow) applying flow at 2 dyn/cm² are shown. 80 cells/experiment were analyzed. n = 3. (H) Soluble fibrinogen binding of CXCL1-stimulated WT and Skap2^−/− neutrophils. n = 4. (I) Adhesion of control or Skap2 knockdown HL60 cells on flow chambers coated with P-selectin, IL-8, and an isotype, anti–Mac-1 activation reporter (CBRM1/5), or anti–Mac-1 (M1/70) antibody. n = 3. (J) Soluble fibrinogen binding of IL-8–stimulated control or Skap2 knockdown HL60 cells. n = 3. (K) Transmigration of WT and Skap2^−/− neutrophils through a TNF-stimulated bEnd.5 cell layer in response to a soluble gradient of CXCL1. n = 3. (L–P) Chemotaxis of WT and Skap2^−/− neutrophils in response to a soluble CXCL1 gradient in vitro. Representative trajectory plots (L), migration velocity (M), accumulated (N) and Euclidean distance (O), and forward migration index of chemotaxing neutrophils (P) are shown. 75 cells/experiment were analyzed. n = 4. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ^#, P < 0.001 versus all other groups; Student's t test (A–C, H–K, and M–P) or one-way ANOVA (E–G). Data are means ± SEM. Ctrl, control. See also Videos 3 and 4.