adherent cells and their position above or below the monolayer (see below); (ii) a single field
was recorded for 15 minutes to follow lymphocyte behaviour; (iii) a further 5 fields were briefly
recorded to analyse the position of lymphocytes above or below the monolayer. Manipulations
and microscopy were carried out inside a Perspex box held at 37°C.
The video recordings were digitised and analysed offline using Image-Pro Plus software
(DataCell Ltd, Finchampstead, UK). The numbers of adherent cells were counted in the video
fields, averaged and then converted to cells per mm2 using the calibrated microscope field
dimensions, and multiplied by the known surface area of the HUVEC to calculate the total
number adherent. This number was divided by the known total number of lymphocytes added, to
obtain the percentage of the lymphocytes that had adhered. Each lymphocyte was classified as
either: (i) phase bright, with round or distorted shape, and adherent to the surface of the HUVEC;
(ii) phase dark and spread, and migrating below the HUVEC. The percentage of adherent
lymphocytes that had transmigrated was calculated at each time, with time zero taken as the end
of the settling period. The migration velocities of phase-dark lymphocytes underneath the
HUVEC were measured by digitising a sequence of images 1min apart for 6min. In each digitised
image, cells were outlined and the position of their centroid determined. Migration velocity
(μm∕min) was the average distance moved by the centroid per minute.
3. Microscopic observation of migration through endothelial cells into collagen gels under static
conditions
HUVEC on collagen gels were washed with M199+BSA to remove residual cytokines,
and purified PBL, PHA stimulated PBL or neutrophils were added for 10min. Non-adherent cells
were removed from the HUVEC by gentle washing with PBSA, and phase-contrast video-