Glass coverslips (No. 1.5, ibidi GmbH) were cleaned by ultrasonication (P60H, Elma Schmidbauer GmbH) in potassium hydroxide and ethanol. A cleaned coverslip was attached to the bottom of a microfluidic chamber (channel width, 3.8 mm; channel height, 0.4 mm; sticky-Slide VI 0.4, ibidi GmbH) and incubated with protein A (10 μg ml−1; Invitrogen) overnight at room temperature. After washing off unbound protein A with HBSS, the chamber was incubated with a recombinant human E-selectin (Sino Biological), which contained the fused C-terminal polyhistidine-tagged Fc region of human immunoglobulin G1 (IgG1) at the C terminus, at 4°C at concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 μg ml−1 for 1 hour. The chamber was then washed with HBSS and blocked with 1% casein solution in phosphate-buffered saline (PBS; Thermo) for 30 min to 1 hour at room temperature. The E-selectin–deposited chamber was used immediately for the cell-rolling assay and SR imaging experiments. The surface densities of the recombinant E-selectin were determined by labeling the surface E-selectin by anti-human CD62E (E-selectin) antibody (HAE-1f clone, BioLegend) conjugated to AF-647 dye. The microfluidic chamber with the E-selectin–coated surface was incubated with the antibody for 1 hour at room temperature. After the incubation, free antibodies were washed off with HBSS. Fluorescence images of the AF-647–conjugated antibodies were recorded using a custom-built wide-field fluorescence microscopy setup (see below). The surface densities of E-selectin were calculated by comparing the fluorescence intensities obtained from the surface and those obtained from single AF-647–conjugated antibodies.

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