Activated CD4+ T cells were resuspended in Ca2+- and Mg2+-free HBS (CMF-HBS), followed by a wash in CMF-HBS with 10 mM EDTA. T cells were washed and resuspended in CMF-HBS and incubated for 20 min at 37°C. Cells were then adjusted to 0.5 mM Ca2+ and 0.5 mM Mg2+ (for control cells), or 1 mM Mn2+ (for Mn2+ treatment), and then added to ICAM-1–coated coverslips (coated with 1 μg/ml overnight at 4°C). After 20 min at 37°C, the cells were washed once in PBS containing Ca2+ and Mg2+ and fixed for 15 min in 3.7% paraformaldehyde (PFA) in PBS. Cells were then blocked and permeabilized in PBS, 0.01% saponin, and 0.05% fish skin gelatin (PSG) for 20 min, followed by 45 min with fluorescent phalloidin (Molecular Probes) in PSG. Cells were washed three times and mounted. For anti-CD3 stimulation, activated CD4+ T cells were washed and resuspended in L15 medium (Gibco) supplemented with glucose (2 mg/ml) and 1% FBS and allowed to interact with coverslips coated with anti-CD3 (10 μg/ml) clone 2C-11 alone or anti-CD3 and ICAM-1 (2 μg/ml). After 20 min at 37°C, the cells were washed once in PBS containing Ca2+ and Mg2+ and fixed for 15 min in 3.7% PFA in PBS. Cells were then blocked and permeabilized in PSG for 20 min, followed by 45 min with fluorescent phalloidin in PSG. Cells were washed three times, mounted, and imaged using a 63× Plan Apo 1.4 NA objective on an Axiovert 200M (Zeiss) with a spinning disc confocal system (UltraVIEW ERS6, PerkinElmer). Three z planes spanning 0.5 μm were collected at the cell-surface interface with an ORCA-ER camera (Hamamatsu). Image analysis was conducted using Velocity v6.3 software. Cells were identified using the “find objects” command, using a low threshold on the actin channel, and total phalloidin staining was quantified per cell on the basis of the integrated pixel intensity.

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