Abs, antibodies; CLEVER-1, common lymphatic endothelial and vascular endothelial receptor-1; CLL, chronic lymphocytic leukemia; ECs, endothelial

cells; FACS, fluorescence-activated cell sorting; FCS, fetal calf serum; GPC, G protein coupled; HGF, hepatocyte growth factor; HSEC, hepatic sinusoidal endothelial cell; ICAM-1, intercellular adhesion molecule-1; IFN-γ, interferon-gamma; IgG, immunoglobulin G; MZL, marginal zone lymphoma; NHL, non-Hodgkin’s lymphoma; PBC, primary biliary cirrhosis; PBS, phosphate-buffered saline; TNF-α, BMS-354825 price tumor necrosis factor alpha; VAP-1, vascular adhesion protein-1; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor. Liver endothelial cells (ECs) were isolated from human liver tissue obtained from explanted livers or donor tissue surplus to surgical requirements, as described previously.4 All tissue was collected from patients in the Liver Unit at the Queen Elizabeth Hospital in Birmingham (Birmingham, UK) with informed consent and under local ethics committee approval. In brief, approximately 30 g of tissue underwent collagenase digestion Silmitasertib manufacturer (0.2% collagenase

type Ia; Sigma-Aldrich, St. Louis, MO). The digested tissue was placed over a 33%/77% Percoll (Amersham Biosciences, GE Healthcare, Little Chalfont, UK) density gradient. ECs were isolated MCE by immunomagnetic selection using antibodies (Abs) against CD31 conjugated to Dynabeads (Invitrogen, Paisley, UK). ECs were then cultured

in medium composed of human endothelial basal growth medium (Invitrogen), 10% AB human serum (HD Supplies, Glasgow, UK), 10 ng/mL of vascular endothelial growth factor (VEGF), and 10 ng/mL of hepatocyte growth factor (HGF) (PeproTech, Peterborough, UK). Cells were grown in collagen-coated culture flasks and were maintained at 37°C in a humidified incubator with 5% CO2 until confluence. Using this protocol, a sufficient number of cells were isolated from either diseased or healthy tissue for use in functional assays. Peripheral blood lymphocytes were isolated as previously described by density-gradient centrifugation over Lympholyte (VH Bio, Gateshead, UK) for 25 minutes at 800×g.13 Harvested lymphocytes were washed in phosphate-buffered saline (PBS) and resuspended in RPMI 1640 with 10% fetal calf serum (FCS). CD4, CD8, and B-cell populations were isolated by using negative immunomagnetic selection kits (Invitrogen). Kits were used as per the manufacturer’s instructions. Highly pure populations of untouched peripheral blood B cells were obtained. Flow cytometry demonstrated greater than 98% expression of CD19 on isolated populations.