Isolation and Purification of Liver Sinusoidal Endothelial Cells

To isolate non-parenchymal cells (NPC), livers were perfused in situ with digestion medium containing collagenase IV (Sigma-Aldrich, Steinheim, Germany) injected into the portal vein, excised and further incubated in the digestion medium. To eliminate parenchymal cells, the single-cell suspension was subjected to a one-step density gradient centrifugation with 26% Nycodenz (Progen Biotechnik, Heidelberg, Germany). LSEC were isolated from NPC by magnetic cell sorting using anti-CD146 antibody (ME-9F1; BioLegend, Fell, Germany) as previously described [19 (link)]. LSEC adhered over night and were subsequently washed in order to remove non-adherent cells resulting in a purity of higher than 99% [20 (link)]. For transmigration assays, CD4+ T cells were isolated from spleen and lymph nodes using anti-CD4 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) to a purity of at least 95%. Untouched CD4+ T cells used for homing assays were isolated by CD4+ T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instruction to a purity of at least 95%.