E-selectin–mediated cell tethering and rolling by resolved human leukocyte gangliosides. (A) Ganglioside resolution. Purified monosialoglycosphingolipids from human granulocytes were resolved on a LiChrosorb NH₂ HPLC column using an acetonitrile/aqueous ammonium bicarbonate gradient as eluent. Only the trailing fractions of the HPLC run are shown (compare with Figure 3; prior fractions did not support E-selectin tethering). Fucosylated species detected by MALDI-TOF MS of the resolved fractions are designated above the chromatogram (dotted lines indicate minor species), with structure abbreviations (n/m) as defined in Figure 1 legend. (B) Tethering (■): Aliquots of each fraction containing from 5 fmol to 20 pmol were adsorbed as a lipid monolayer on Petri dishes in a discrete 5-mm diameter circular area. CHO cells stably expressing E-selectin were perfused over the adsorbed lipid at 1 dyne/cm² for a 5-minute period, during which tethered cells were quantified. The minimal ganglioside concentration resulting in at least 100 tethers/mm² was determined. Potency is expressed as the inverse of the minimal effective concentration. Rolling (): Aliquots containing 100 to 300 fmol GSL were adsorbed and rolling velocities (μm/s) of CHO cells stably expressing E-selectin were determined as the displacement of the centroid of the cell over a 5-second interval. The means plus or minus SEM of 6 to 34 cells are shown.