Neutrophil function tests. (A) Chemotaxis upon stimulation with C5a (10 nM), IL-8 (10 nM), PAF (100 nM), and fMLF (30 nM) measured using 3-µm pore–sized filters (mean + range, n = 2 of the index patient). (B) MKL1-deficient neutrophils show defective spreading behavior upon a 10-minute stimulation with fMLF (10 nM). Neutrophil spreading is quantified as the percentage of increase in size (mean + SEM, n = 1, 13-19 cells counted per condition). (C) Adhesion as percentage of total input upon stimulation with fMLF (1 µM), C5a (10 nM), PMA (100 ng/mL), or dithiothreitol (DTT, 1 mM) (mean + range, n = 2 of the index case). (D) Transendothelial migratory events were quantified and normalized to control neutrophils. Neutrophils from patient 2 were used (mean + standard error of the mean; n = 1, duplicate). (E) Protease release upon stimulation with fMLF (1 µM) or CytoB:fMLF (5 µg/mL:1 µM) or in a Triton X-100 lysate (TX-100) was determined by DQ Green BSA (mean + range, n = 3, both patients). (F) Extracellular reactive oxygen species production in response to opsonized E coli (0.25 × 10⁹/mL), zymosan (1 mg/mL), serum-treated zymosan (STZ; 1 mg/mL), PMA (100 ng/mL), or PAF/fMLF (1 µM/1 µM) was measured using an Amplex Red assay (mean + range, n = 2, index patient).