Direct observations of the kinetics of migrating T-cells suggest active retention by endothelial cells with continual bidirectional migration

Video recordings were made of a series of microscope fields along the centreline of the flow
channel after 2 and 11min of washout, and between these times a single field was recorded.

Video recordings were analysed essentially as above, except that lymphocytes adherent to
the surface of HUVEC could be classified as either rolling adherent (spherical cells moving over
the surface much slower than free-flowing cells) or stationary adherent (typically with distorted
shape and actually migrating slowly on the surface). Phase-dark transmigrated cells could be
counted for either substrate, but when observing filters in the flow chamber, recordings were also
made of the underside of the filter where a few cells might be found. This was achieved by
focussing the microscope stage up and down 10μm (i.e., the filter thickness). The sum of the
numbers adherent in all categories was divided by the number perfused during the bolus to obtain
total PBL adhesion as % of cells perfused.

Statistical analysis

Effects of multiple treatments were tested using analysis of variance (ANOVA), followed
by comparison to control by Dunnett test. Single treatments were compared to controls by paired
t-test.

Results

Kinetics of lymphocyte migration through endothelial cells and 3μm-pore filters

Settling of lymphocytes onto endothelial cell/filter constructs and quantification of the
number collected from the back has been widely used to assess 'transendothelial' migration. In
initial experiments with unstimulated or TNF-treated HUVEC, we found that few PBL migrated

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