HPK1-deficient PMNs showed normal affinity regulation of Mac-1, but defective CXCL1-mediated upregulation of LFA-1 affinity. (A) Flow cytometric analysis of cell-surface expression of Gr-1, CD18, CD11a, and CD11b. Isolated murine PMNs were stimulated with CXCL1 (100 ng/mL), TNF-α (100 ng/mL), N-formyl-methyl-leucyl-phenylalanine (10 µM), or left unstimulated for 20 minutes at 37°C. Histograms are representative of 3 independent experiments. (B) Binding of soluble fibrinogen to isolated murine PMNs measured by flow cytometry. PMNs were stimulated with CXCL1 (100 ng/mL), Mn²⁺ (3 mM), or left unstimulated. Bar chart (left) shows percentage of cells with positive fibrinogen binding, calculated by using a threshold that defined 95% of EDTA-treated (2 mM) PMNs as negative. Representative fluorescence-activated cell sorting (FACS) histograms are shown on the right. n = 6. (C) LFA-1–specific binding of soluble ICAM-1/Fc to isolated murine PMNs measured by flow cytometry. PMNs were preincubated with anti–Mac-1 blocking antibody to prevent Mac-1 binding of ICAM-1/Fc and were stimulated with CXCL1 (100 ng/mL), Mn²⁺ (5 mM), or left unstimulated. Bar chart (left) indicates percentage of cells with positive LFA-1–specific ICAM-1/Fc binding, calculated by using a threshold that defined 95% of PMNs treated with CXCL1 and anti–LFA-1 antibody as negative. Similar results were obtained with 3 mM Mn²⁺. Representative FACS histograms are shown on the right. n = 4 (unstimulated, CXCL1); n = 3 (Mn²⁺). Diagrams show mean ± SD; *P < .05; n.s. = not significant.