Anti human cd34 fitc

The purified CD34⁺cells were seeded onto 6-well plates (2×10⁶ cells/well) coated with 3% recombinant human gelatin (Prospec-Tany TechnoGene Ltd., Rehovot, Israel) and cultivated in endothelial growth medium:EBM-2 (Lonza Group Ltd., Basel, Switzerland) containing 10% heat-inactivated fetal calf serum (FCS; Invitrogen; Thermo Fisher Scientific, Inc.), 10% horse serum (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 10 ng/ml stem-cell growth factor (TEBU GmbH, Frankfurt, Germany), 2 mM L-glutamine and 5 ng/ml VEGF (Sigma- Aldrich; Merck KGaA) and 10 ng/ml SCGF (Sigma-Aldrich; Merck KGaA). Cells were maintained at 37°C in an atmosphere containing 5% CO₂. Nonadherent cells were removed after 48 h, and adherent cells were resuspended. A total of 25% of the cells were incubated for 30 min at room temperature, with: Anti-human CD34-FITC (1:50; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), anti-human CD133-phycoerythrin (1:50; Miltenyi Biotec GmbH), mouse anti-human VEGFR2-allophycocyanin (APC) primary (1:50) and rabbit anti-mouse-APC secondary (1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) antibodies, and subsequently analyzed by flow cytometry as previously described (15 (link)). Cell suspensions were prepared in PBS containing 1% (vol/vol) FCS, using 2% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) to block non-specific antigens on the cells.