Migratory blockade by CD177 ligation reflects enhanced adherence. (A) The proportion of neutrophils adherent to the 3-µm transwell filters that were CD177^pos was quantitated by confocal microscopy in the presence of isotype control or αCD177. Anti-CD177 increased the proportion of adherent CD177^pos cells. (B) Neutrophils were exposed to a migratory stimulus (LTB₄ 100 nM) across a barrier with pores too small to permit migration (0.4 µm), and the proportion of adherent neutrophils was assessed in the presence of isotype or αCD177. Anti-CD177 increased adherence to the membrane. Data for panels A and B represent triplicate wells per condition, representative of at least 2 to 3 similar experiments. (C) Neutrophils were incubated on glass coverslips in the presence of LTB₄ 100 nM for 15 min and washed, and CD177^pos cells were characterized by paired differential interference contrast and immunofluorescence microscopy as attached (rounded shape with or without a trailing tail) or spreading (“halo” staining of CD177, lamellar protrusions, or both). Scale bars measure 10 µm. αCD177 markedly induced spreading morphology (D) and increased the cellular area of CD177^pos neutrophils (E). (F) Treatment with anti-CD177 increases the fraction of shear stress resistant neutrophils. Data reflect 2 replicates enumerating 90 to 110 cells each (C-D), 25 to 30 cells each (E), and 3 independent experiments performed in duplicate, each replicate analyzing 10 to 50 cells per field of view (F). Means ± SEMs. *P < .05; **P < .01; ****P < .0001; by 2-way ANOVA (A,D) or unpaired t test (B,E,F). DIC, differential interference contrast. Ctl, control.