125I-α-Gal released from anti-ICAM/125I-α-Gal NCs was separated from the particle-bound fraction by centrifugation after incubation for varying periods of time in (a) 1% BSA in PBS buffer at 4°C or 37°C; (b) 1% BSA in PBS, HUVEC medium, or FBS at 37°C and pH 7.4; (c) HUVEC medium at 37°C and either pH 7.4 or pH 4.5; and (d) HUVEC medium at 37°C and pH 4.5 in the absence or presence of NBD-Gb3. Data are mean±SEM (n≥3). * compares each time point to time 0 within the same condition. # compares (for each time point): (a) 4°C to 37°C, (b) HUVEC medium and FBS to BSA, (c) pH 4.5 to pH 7.4, and (d) Gb3 to non-Gb3. * and # are p≤0.05; ** and ## are p≤0.01; *** and ### are p≤0.001, by Student’s t-test. Statistical significance for time 0–8 h are shown only in the left panel and time 24–72 h only in the right panel.