HPK1 was important for LFA-1–mediated adhesion strengthening and spreading of PMN under flow conditions in vitro. For flow chamber assays, isolated murine PMNs were treated with an isotype control antibody (30 µg/mL) or an anti–Mac-1 blocking antibody (30 µg/mL) and seeded into flow chambers coated with P-selectin (10 µg/mL), ICAM-1 (12.5 µg/mL), and CXCL1 (5 µg/mL). After 10 minutes of static conditions, flow was applied as indicated. (A) Detachment assay of adherent murine PMNs in response to gradually increasing shear stress (0.1 to 8.0 dyne/cm²) at intervals of 90 seconds. Adhesion strengthening is represented by the percentage of nondetaching cells compared with initially adherent cells at 0.1 dyne/cm² (100%). n = 3 independent experiments with a total of 315 (HPK1^+/+ isotype control), 281 (HPK1^−/− isotype control), 266 (HPK1^+/+ anti–Mac-1), and 235 (HPK1^−/− anti–Mac-1) analyzed cells. (B) Spreading of adherent PMNs after 5 minutes of constant flow (1 dyne/cm²), quantified by the measurement of cell area (left panel). Representative microscopic images (right panel) are shown. Bar = 10 µm. n = 3 (isotype control); n = 4 (anti–Mac-1) independent experiments with a total of 221 (HPK1^+/+ isotype control), 318 (HPK1^−/− isotype control), 344 (HPK1^+/+ anti–Mac-1), and 460 (HPK1^−/− anti–Mac-1) analyzed cells. Diagrams (A,B) show mean ± SD; *P < .05 for HPK1^+/+ values versus corresponding HPK1^−/− values.