ZAP-70 is not involved in T-cell arrest on inflammatory endothelium or in CXCL12-induced actin polymerization.

(A) Jurkat and P116 cells were labeled with different fluorescent dyes, washed, mixed, and perfused through a flow chamber at a flow rate of 40 mL/h on the monolayer of endothelial cells previously activated by TNF-α for 18 hours or left untreated. T-cell arrest was quantified by measuring the total number of firmly adherent cells arrested on 6 independent fields chosen at random. (B) A similar experiment was performed on chambers coated with recombinant soluble VCAM-1 (rsVCAM-1) with Jurkat and P116 cells that were previously incubated with a blocking anti–VLA-4 mAb or were left untreated. (C) Jurkat and P116 cells were incubated for the indicated times with CXCL12 (50 ng/mL). Cells were then fixed and permeabilized in the presence of 0.05% saponin, and FITC–phalloidin was added for 30 minutes before the final wash. Cells were then analyzed by flow cytometry. Results are expressed as the ratio of the mean fluorescence intensity (MFI) of stimulated cells to the MFI of unstimulated cells.