Pe conjugated mouse anti human cd34 mab

Human umbilical cord blood samples (70–100 ml each; n = 10) were collected in sterile blood packs containing citrate–dextrose solution as the anticoagulant. After a Ficoll Hypaque (density 1077 g/cm³; GE Healthcare, Norway) density gradient centrifugation, the MNCs were isolated, washed twice, and suspended in sterile PBS with 0.5% BSA and 2 mM EDTA. CD34⁺ cells were separated from MNCs by a magnetic bead separation method (Miltenyi Biotec, Germany). In brief, the total MNCs were labeled with mouse anti human CD34 mAb coupled with microbeads (MACS). The bead-positive cells (MNC-CD34⁺) were enriched on positive-selection columns set in a magnetic field. Flow-cytometric analyses of purified cells using a PE-conjugated mouse anti human CD34 mAb (BD Biosciences, USA) showed that 94.1 ± 5.3% of the selected cells were positive for CD34.

Adherent cells were first detached using trypsin and collected together with suspended cells in PBS. The cells were then incubated with antibodies or the IgG isotype control for 30 min at room temperature. The antibodies used were PE-conjugated mouse anti-human CD34 mAb, APC-conjugated mouse anti-human CD133 mAb, PE-conjugated mouse anti-human VEGFR-2, APC-conjugated mouse anti-human CD31 mAb, FITC-conjugated mouse anti-human CD144 mAb, PE-conjugated mouse anti-human CD45 mAb, and PE-conjugated mouse anti-human CD14 mAb (BD Biosciences). Data were collected on a FACSVerse flow cytometer (BD Biosciences) and analyzed with FlowJo.