Binding of aICAM-1 L to non-activated and TNF α-activated bEnd.5 cells. (a) Cellular NIR fluorescence intensity of non-activated and TNFα-activated bEnd.5 cells after 2 h incubation at 37 °C with aICAM-1 L, prepared using different Ab:SATA ratios, quantified with FACS. Data were corrected for cellular fluorescence after incubation with L and reflect the increase in fluorescence by aICAM-1 conjugation. Application of IgG L did not lead to an increase in fluorescence intensity (not shown). * = p < 0.05 vs. all groups, ** = p < 0.05 vs. IgG L, ANOVA with Bonferroni correction. n = 3-4. (b) CLSM images illustrating the cellular distribution of aICAM-1 L (Ab:SATA = 1:80) and IgG L (Ab:SATA = 1:80) (red). The cell membrane was labeled with CD31 (green) and cell nuclei were counterstained with DAPI (blue). Laser power 488 nm: 25%, 780 nm: 4%, 633 nm, IgG L: 50%, aICAM-1 L/-TNFα: 10% and aICAM-1 L/+TNFα: 5%. Scale bar = 50 μm.