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

gentamycin, 1μg∕ml hydrocortisone (all from Sigma) and 2.5μg∕ml amphotericin B (Gibco
Invitrogen Compounds). Primary HUVEC were dissociated using trypsin/EDTA (Sigma) and
seeded on either six-well tissue culture plates (Falcon; Becton Dickinson Labware, NJ, USA),
uncoated low-density 3.0μm pore polycarbonate Transwell filters (which were placed in
matching plates; BD Pharmingen, Oxford, UK), glass chamber slides (Lab-tek, Nalge Nunc
International, Naperville, IL) or collagen gels (see below). Seeding density was chosen to yield
confluent monolayers within 24h. Tumour necrosis factor-alpha (TNF; 100U/ml; Sigma) and/or
interferon gamma (IFN; 10ng/ml; Peprotech Inc., London, UK) were added to confluent
monolayers for 4 or 24h before the assay with neutrophils or lymphocytes respectively.

To form collagen gels, type 1 collagen dissolved in 0.6% acetic acid (2.15mg/ml; First Link Ltd,
West Midlands, UK) was mixed with 10xM199 and FCS (1.66ml, 0.1ml and 0.34ml
respectively). The pH was neutralised by addition of 0.15ml 1N NaOH, and 1ml was dispensed
into a 6-well plate and allowed to gel at 37°C. The gel was then equilibrated with HUVEC
culture medium for 48h before seeding and culture with HUVEC as above. In some experiments,
CXCL10 (IP-10) or CXCL12 (SDF1-α) (80 or 800ng/ml; Peprotech) was added to the collagen
after neutralisation with NaOH. The gel was polymerised as above and equilibrated with
HUVEC culture medium containing chemokine at the same concentration.

Analysis of lymphocyte migration

1. Migration through endothelial cells on Transwell filters under static conditions

Lymphocyte migration was assessed using 24-well format Transwell filters. HUVEC
were washed to remove residual cytokines, fresh M199+BSA was placed in the lower chamber

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