Lab-Tek 8 chamber slides (Thermo Fisher Scientific) were coated with ICAM-1 (2 μg/ml) overnight at 4°C. Activated CD4+ T cells were washed and resuspended in L15 medium (Gibco) supplemented with glucose (2 mg/ml) and 1% FBS. T cells were then added to the chambers, incubated for 20 min, gently washed to remove all unbound cells, and imaged using a 10× phase objective at 37°C on a Zeiss Axiovert 200M inverted microscope equipped with an MS-2000 automatic stage (Applied Scientific Instruments) and a Roper Scientific electron-multiplying CCD camera. Time-lapse imaging was performed using SlideBook 6 software (Intelligent Imaging Innovations Inc.), collecting one image every 30 s for 10 min total. For drug treatments, the indicated drug was added and preincubated 5 min before imaging. Movies were exported into ImageJ, and cells were tracked using the manual tracking plugin. Directionality was calculated in Excel by dividing net displacement by total track length for each cell. Cells were scored as motile if they showed a polarized morphology and migrated at least three cell diameters. For higher-magnification movies, cells were imaged every 5 s for 3 to 5 min using a 63× objective.

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