sVCAM-1 binding to isolated peripheral blood lymphocytes.
Human peripheral blood lymphoid cells were isolated as described in “Materials and Methods.” Cells were resuspended in Tyrode's buffer containing 1 mmol/L CaCl2, 1 mmol/L MgCl2, and 0.1% BSA and incubated for 30 minutes at room temperature in the absence of stimulation (upper panels) or in the presence of PMA (50 ng/mL) (lower panels). In the presence or absence of the function blocking anti-α4 mAb HP2/1, 7 Ig-domain VCAM-Ig (20 μg/mL) was added to the cells, and the mixture was incubated for 30 minutes at room temperature. After washing, the cells were stained with FITC-anti-CD3 mAb (HIT3a). At the same time, they were stained with PE-conjugated donkey antihuman IgG to detect the VCAM-Ig fusion protein as described in “Materials and Methods.” The percentage of cells in quadrants is indicated. Results are representative of 3 separate experiments.
