and PBL were added to the upper chamber. The lymphocytes were allowed to settle, adhere and
migrate through HUVEC at 37oC in a CO2 incubator for the desired period. Migration was
stopped at the chosen time by transferring the filter into a fresh well, leaving the transmigrated
cells in the original lower chamber. The lymphocytes suspended in the upper chamber were
removed, and pooled with cells obtained when the filter was washed twice. These cells were
taken to represent non-adherent lymphocytes. The non-adherent and transmigrated cells were
counted using a Coulter Multisizer II (Coulter Electronics Ltd, Essex, UK). From the known
number of added lymphocytes, the percentage of lymphocytes that adhered, and the percentage of
lymphocytes that transmigrated were calculated.
In some experiments, the surface phenotypes of adherent and transmigrated lymphocytes
were assessed by flow cytometry. Freshly isolated, non-adherent (upper chamber) or
transmigrated (lower chamber) lymphocytes were labelled with anti-CD4-PE or anti-CD8-FITC
(Becton Dickinson, Oxford, UK) for 30min on ice. Fixed volume counts for positively labelled
cells were made using a Coulter XL flow cytometer and analysed using WinMDI. In this way,
we calculated the percentage adhesion and transmigration for CD4+ and CD8+ T-cell subsets.
2. Microscopic observation of migration through endothelial cells under static conditions
Adhesion and transmigration were assessed by direct microscopic observation as
previously described [26]. HUVEC in 6-well plates were washed with PBSA to remove residual
cytokines and purified PBL, CD3+ or CD4+ T-cells were added for 5min. Non-adherent cells
were removed from the HUVEC by gentle washing with PBSA (which took 2min) and video
recordings of the endothelial surface were made using phase contrast videomicroscopy as
follows: (i) 5 fields were briefly recorded immediately after washing to analyse number of