Human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex (Germany) and cultured in endothelial basal medium (EBM) supplemented with 1 μg/mL hydrocortisone, 12 μg/mL bovine brain extract, 50 μg/mL gentamicin, 50 ng/mL amphotericin-B, 10 ng/mL epidermal growth factor and 10% fetal calf serum until the third passage. For silencing human Del-1 we used ON-TARGETplus SMARTPool siRNAs and control siRNA from Dharmacon. For transfection we used Lipofectamine RNAiMAX (Invitrogen, Germany) according to the manufacturer’s protocol.
Peripheral blood-derived mononuclear cells (MNCs) were isolated by density-gradient centrifugation with Ficoll (Biochrom AG, Germany) from buffy coats from healthy human individuals as described previously. For homing experiments, bone marrow (BM)-derived Lin− progenitor cells were purified from WT mice by negative selection using a cocktail of biotinylated antibodies to lineage markers (Lineage cell depletion kit, mouse, Milteny Biotec, Bergisch-Gladbach, Germany) for 10 min at 4°C followed by anti-biotin microbeads for 15 min (Miltenyi Biotec) as described elsewhere (8 (link), 32 (link)).
Peripheral blood-derived mononuclear cells (MNCs) were isolated by density-gradient centrifugation with Ficoll (Biochrom AG, Germany) from buffy coats from healthy human individuals as described previously. For homing experiments, bone marrow (BM)-derived Lin− progenitor cells were purified from WT mice by negative selection using a cocktail of biotinylated antibodies to lineage markers (Lineage cell depletion kit, mouse, Milteny Biotec, Bergisch-Gladbach, Germany) for 10 min at 4°C followed by anti-biotin microbeads for 15 min (Miltenyi Biotec) as described elsewhere (8 (link), 32 (link)).
