The cell-rolling assay was performed at room temperature using a microfluidics chamber sticky-Slide VI 0.4 attached to the E-selectin–deposited glass coverslip. The inlet and outlet of the chamber were connected to a 0.8-mm silicon tubing (ibidi GmbH) using male Luer connectors (ibidi GmbH). The end of the inlet tube was placed in a rolling buffer [HBSS containing 1% human serum albumin (Sigma) and 1 mM CaCl2 (Sigma)], and the end of the outlet tube was connected to a programmable syringe pump (PHD ULTRA, Harvard Apparatus) using a female Luer Lock connector (ibidi GmbH). To equilibrate the flow path, we allowed the rolling buffer to flow into the chamber for 90 s prior to introducing the KG1a cells. Then, 106 ml−1 KG1a cells suspended in the rolling buffer were autoperfused for 20 s using a syringe pump. After a brief stop in flow for 10 to 20 s to allow the cells to settle down and interact with the E-selectins on the surface, we resumed flow of the cell suspension for another 20 to 30 s. Then, the chamber was washed with the rolling buffer for 90 s, during which we recorded a video of the cells rolling on the E-selectin–coated surface of the chamber. Multiple videos were recorded with a time interval of 105 to 115 s for the analysis of time-dependent rolling behaviors. The rolling experiment was conducted at wall shear stresses (W) of 0.25, 0.5, 1, 2, and 4 dyne cm−2. The cell-rolling behavior was captured by mounting the microfluidic chamber on an inverted optical microscope (CXK41, Olympus) equipped with a 20× objective (LCAch N 20X, Olympus) and by recording transmitted light images at video rate using a CCD camera (XC10, Olympus) using CellSens software (Olympus). Transmitted optical microscopy images of the KG1a cells were also recorded using an Olympus IX71 inverted optical microscope equipped with a high numerical aperture (NA) objective (UAPON 100XOTIRF, Olympus) and an EMCCD camera (iXon3 897, Andor Technology).

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