the static assay, and again, velocity tended to be faster in the presence of IFN (Table 1C).
We checked whether multiple transits were observed under flow as well as in the static
assay. In fact, the proportions of cells undergoing at least one transit, or undergoing multiple
transits in a six-minute period were similar, if a little higher, in the presence of flow (Table 1C).
Overall, the average intervals between transits were nearly identical to the values obtained in the
static assay (Table 1). Again, values for these variables were similar for the different cytokine
treatments.
Lymphocyte migration through endothelial cells and into collagen gels
The above findings suggested that under static or flow conditions, lymphocytes quickly
crossed endothelial monolayers, but were reluctant to move on from the subendothelial space,
and that this might have been linked to repeated migration back and forth. However, even the
filters represent a solid barrier to migration over most of their surface, and so we decided to
observe migration out of the subendothelium into collagen gels over prolonged periods. First, we
analysed neutrophil migration through TNF-treated HUVEC into gels, since we had characterised
neutrophils previously in all the other models used here [e.g., 21, 22, 26]. Figure 7A shows
changes in the distribution of neutrophils above and just below the EC, and in the gel over time.
After 15 minutes, a high proportion of adherent neutrophils had penetrated the monolayer and
were visible, phase-dark just below it. By 1 hour, a few more cells had migrated under the EC,
but nearly all of the transmigrated cells were now found in the gel. The cells moved further into
the gel by 3 hours, and by 24 hours, the neutrophils were essentially evenly distributed
throughout the depth of the gel. The lymphocytes were much less efficient in entering the gels
(Figure 7B). A significant proportion of adherent cells had migrated through the endothelial