Values were converted into biomolecule site densities using an established fluorescenceradioactivity correlation for AbMole Nitroprusside disodium dihydrate P-selectin and E-selectin. It is well established that PSGL-1 is the predominant and most well-characterized binding partner of P-selectin. However, we wanted to validate the specific targeting of PSGL-1 by bromelain as an effector of rolling and tethering on P-selectin substrates and therefore performed an additional control experiment. This experiment utilized neutrophil-like HL-60 promyelocytes, which also tether and roll on P-selectin surfaces via PSGL-1, and a monoclonal anti-PSGL-1 blocking antibody with and without bromelain treatment. As shown in Figure 4, AbMole Miglitol treatment with the blocking antibody alone led to a marked decrease in the number of tethering events on both “high” and “low” P-selectin substrates. Importantly, HL-60 cells that were treated with bromelain after being blocked with the antibody did not show a further reduction in substrate interaction, confirming the specific action of bromelain on PSGL-1 as it leads to reduced interactions with P-selectin surfaces. P-selectin is the primary molecular player that controls initial neutrophil interactions with the inflamed endothelium; however E-selectin is also known to play a key role in mediating leukocyte rolling. PSGL-1 is a ligand for both P- and E-selectin and therefore we wanted to see whether bromelain treatment similarly abolished neutrophil interactions on E-selectin. Isolated human neutrophils were flowed over substrates presenting two different site densities of immobilized E-selectin. In contrast to experiments on Pselectin-presenting substrates, bromelain treatment had no significant effect on neutrophil tethering to immobilized E-selectin. The cutaneous lymphocyte antigen glycan moiety pendant on PSGL-1 and other ligands, plays a role in leukocyte tethering and rolling mediated by E-selectin. To see if neutrophil interactions with immobilized E-selectin could be altered by blocking CLA on all E-selectin ligands, we incubated neutrophils with saturating levels of HECA-452, a widely used mAb that targets CLA. Not surprisingly, this blocking antibody reduced tethering to immobilized E-selectin, though the effect was not as significant as that for the anti-PSGL-1 blocking on P-selectin substrates. The incomplete inhibition of tethering on E-selectin after anti-CLA blocking reflects the fact that E-selectin, unlike P-selectin, has a diverse range of potential binding partners and thus simply blocking one binding epitope does not abolish all interactions, a finding consistent with the literature. Overall, neutrophil flow assays with E-selectinpresenting substrates reveal that bromelain does not proteolytically abolish all interactions between neutrophils and E-selectin.