Ninety-six–well plates (MaxiSorp, Thermo Fisher Scientific) were coated with mouse ICAM-1 (1 μg/ml) in phosphate-buffered saline (PBS) overnight at 4°C. Plates were then washed three times with PBS, blocked with 1% bovine serum albumin (BSA) in PBS for 1 hour at room temperature, and washed twice with PBS. Activated CD4+ T cells were used on days 5 to 7 after initial isolation. To prepare the cells, they were first labeled with calcein-AM (Thermo Fisher Scientific) at a final concentration of 2.5 μM for 30 min at 37°C in serum-free DMEM. Cells were then washed and resuspended in T cell complete medium and incubated at 37°C for 30 min. For Mn2+ treatment, the cells were then washed in Hepes-buffered saline (HBS), 1% BSA, 10 mM EDTA; washed again in HBS, 1% BSA; and resuspended in HBS, 2.5% BSA with 1 mM Mn2+. Otherwise, cells were resuspended in 2.5% BSA in PBS (with Ca2+ and Mg2+). In each case, 1 × 105 cells were added to each well on ice in triplicate. After a 20-min incubation, the plate was read on a Bio-Tek Synergy HT fluorescence plate reader to obtain the baseline measurements representing “maximum adhesion” per well. Where indicated, PMA was added to a final concentration of 10 ng/ml and the plate was incubated at 37°C for 10 min, washed, and read again. The plate was washed and read a total of two to four times until the signal from the unstimulated control was stable. To calculate the percent adhesion, the fluorescence per well after washes was divided by the initial maximum adhesion fluorescence reading per well. Background was subtracted using values obtained from empty wells.

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