Glycomimetic antagonism of selectin mediated β2-integrin activation. (A) PMN rolling on a substrate of E-selectin in the presence of vehicle control, Rivipansel (6.5 μM), or GSnP-6 (12 μM) was dynamically imaged using qDF to detect L-selectin (AF488 anti-human DREG55) and PSGL-1 (PE anti-human PL-1) engagement in the plane of adhesive contact. (B) L-selectin (FITC DREG-55) and (C) PSGL-1 (PE PL-2) receptor cluster area and frequency were determined and reported as mean ± SEM (n = 3 separate experiments; **P < .01 compared with vehicle). (D) PMNs imaged by TIRF were pretreated with a membrane dye (DiI) and HA β2-integrin reporter antibody (mAb24) and perfused over E/I substrates treated with vehicle, Rivipansel (6.5 μM), or GSnP-6 (12 μM). (E) HA β2-integrin cluster number (MFI > 10 pixels above background more than ∼0.1 μm in area) in plane of contact were quantified using qDF in real time and compared between fixed cells that had rolled to arrest between the 3 conditions (n = 3 separate experiments; **P < .01 compared with vehicle).
