High affinity binding of a sVCAM-Ig fusion protein and soluble FN to 4β1 integrins on Jurkat cells.

Cells were resuspended in DMEM containing 0.1% BSA or in a Tyrode's buffer containing 1 mmol/L MnCl₂ and 0.1% BSA (Mn⁺⁺). The indicated concentrations of VCAM-Ig (A) or FITC-FN (B) were added, and the mixtures were incubated for 30 minutes at room temperature. ○, Mn⁺⁺ added; □, no addition. Where indicated by • and ▪, function-blocking anti-α4 mAb HP2/1 was added before the soluble ligand. VCAM-Ig binding was evaluated with FITC-conjugated donkey antihuman IgG using flow cytometry as described in “Materials and Methods” and expressed as geometric mean fluorescence intensity (MFI). (C) Jurkat cells were resuspended in either DMEM containing 0.1% BSA (solid line), DMEM with the addition of 5 mmol/L EDTA (shaded line), or Tyrode's buffer containing 1 mmol/L MnCl₂(dotted line). VCAM-Ig containing only the first 2 N-terminal or all 7 Ig domains (20 μg/mL) was added to the cells, and the mixtures were incubated at room temperature for 30 minutes. Results are representative of 3 separate experiments.