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



Methods

Isolation of human peripheral blood lymphocytes (PBL), T cells and neutrophils
Venous blood from healthy individuals was collected in EDTA tubes (Sarstedt, Leicester, UK).
Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation of blood on
histopaque 1077, and PBL were prepared by panning of PBMC on culture plastic to remove
monocytes [23]. In some experiments, to obtain activated T-cells, PBMC were cultured for 7
days in the presence of 10
μg∕ml phytohaemagglutinin (PHA; Sigma). PHA initially induced
clumping and proliferation of lymphocytes, but by day 7 cells had dispersed and were spherical
and smaller than freshly isolated PBL (judged by Coulter Counter volume distribution). In other
experiments, T-cells (CD3+) or CD4+ T-cells were purified from PBMC by negative selection
using magnetic Dynabeads(Dynal, Wirral, U.K.) and a cocktail of monoclonal antibodies to
remove cells bearing CD19, CD11b (HIB19 and ICRF44 respectively, both from BD
Pharmingen, UK), CD14, CD16 (RM052 and 3G8 respectively, Beckman Coutler, UK) and, in
the case of CD4+ T cell selection, CD8 (OKT8, eBioscience, UK) [24]. Isolated cells were
washed, counted, and adjusted to a final concentration of 2x106/ml in phosphate buffered saline
containing Ca2+ and Mg2+ or Medium 199 (Gibco Invitrogen Compounds, Paisley, Scotland)
supplemented with 0.15% bovine serum albumin (Sigma-Aldrich, Poole, UK) (PBSA or
M199BSA respectively). On a few occasions, neutrophils were isolated using a two-step density
gradient as described [20-22] and suspended at 2x106/ml in M199BSA.

Isolation and culture of endothelial cells

HUVEC were isolated from umbilical cords as previously described [25] and cultured in
M199 supplemented with 20% fetal calf serum (FCS), 10ng/ml epidermal growth factor, 35L.lg 'ml



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