microscope recording were made 0.25h, 1h, 3h and 24h after the original addition of leukocytes.
Five video-fields were recorded. In each field, images were first recorded at the endothelial
surface, and then recordings were made as the microscope was focussed gradually down in 50μm
steps. Cells visible with the endothelial monolayer were counted and divided into those which
were phase bright (above EC) and those which were phase dark (just below EC). Cells within
each 50μm step were counted as they came into focus; these cells were typically irregular in
shape and phase bright. The focal depth of the gels was approximately 300μm. After averaging
counts in the 5 fields, data were expressed as the percentage of the adherent cells in each vertical
region. On some occasions, a single field at the endothelial surface was recorded for 5 minutes to
analyse migratory behaviour of the leukocytes. All manipulations of gels and microscopy were
carried out at 37°C.
4. Microscopic observation of migration through endothelial cells under conditions of flow
Filters coated with HUVEC were cut from the Transwell holders and placed on a
75x25mm coverslip. Alternatively, the bases of chamber slides coated with HUVEC were freed
from the fluid reservoirs attached to their surface. The coverslips or slides were incorporated into
a parallel-plate flow chamber and attached to a perfusion system mounted on the stage of a
phase-contrast videomicroscope enclosed in a Perspex chamber at 37oC, as described [21, 26].
The flow channel dimensions were 20 x 4 x 0.13mm (length x width x depth) for the filters, and
50 x 10 x 0.25 mm for the chamber slides. At one end, they were connected to a Harvard
withdrawal syringe pump which delivered flow at a rate equivalent to a wall shear stress of 0.1Pa.
At the other end, they were connected to an electronic switching valve (Lee Products, Gerards
Cross, UK) which selected flow from two reservoirs, containing PBL in PBSA or cell-free PBSA.
A four-minute bolus of PBL was perfused over the HUVEC followed by cell-free wash buffer.