E selectin

Briefly, a straight microchannel (1 cm length by 400 μm width by 60 μm height) was coated with E-selectin (50 μg/mL, Peprotech) for 1 h at 4 °C and blocked with 0.5% BSA in PBS for 30 min at room temperature. After blocking, the channel was connected to a syringe loaded with 0.1% BSA in PBS to prime and wash away excess E-selectin in the channel. For rolling assay, CaCl₂ (20 μM, Sigma-Aldrich) was added to the DFF-purified neutrophils to facilitate the calcium-dependent interactions of neutrophil binding and rolling on E-selectin. ~10–20 μL of DFF-purified neutrophils (~10⁶cells/mL) was loaded at the inlet reservoir and the syringe was set to withdraw at 2.6 μL/min (~2 dynecm⁻²) to initiate neutrophil rolling. Phase contrast image was captured for 30 s at the centre of the microchannel every 0.5 s interval (total of 61 frames) at 20× magnification using MetaMorph software (Molecular Devices). To induce inflammation and disease conditions, DFF-purified neutrophils were also treated with tumor necrosis factor alpha (TNF-α, 10 ng/mL, Peprotech), PMA (2 nM, Sigma-Aldrich) or D-glucose (30 mM, Sigma-Aldrich) for 30 min at room temperature. The neutrophils were then washed at 1000× g for 4 min and resuspended to a concentration of ~10⁶cells/mL with 20 μM CaCl₂ for rolling assay.