Anti cd34 pe clone qbend 10

For determining the percentage of tip cells in cell cultures, cells were fixed in 4% paraformaldehyde in PBS for 10 min and incubated with anti-CD34-phycoerythrin (1:50; anti-CD34-PE; clone QBend-10, Thermo Scientific, Waltham, MA, USA) for 30 min at room temperature. Tip cells were identified as CD34-positive cells using a FACSCalibur (Beckton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo 6.4.7 software (Tree Star, San Carlos, CA, USA). The FITC channel was used to detect autofluorescence. Non-stained and non-treated cells were used as negative controls. For cell sorting experiments, cells were sorted on the basis of CD34 expression (range between 5–20%) with anti-CD34-PE on a Sony SH800Z cell sorter (Sony Biotechnology, Tokyo, Japan). After cell sorting, CD34⁻ hMVECs and HUVECs were put back in culture for respectively 6 or 24 h, cells were fixed, stained and analyzed using flow cytometry as described above.

The percentage of tip cells in cell cultures was determined as described earlier^{35 (link)}. Briefly, HUVECs were reverse transfected with siRNA and cells were harvested by TrypLE (Gibco) treatment at 72 h after siRNA transfection. Cells were incubated with CD34-phycoerythrin (1:50; anti-CD34-PE; clone QBend-10; Thermo Scientific, Waltham, MA, USA) in PBS for 30 min at room temperature. Tip cells were identified as CD34⁺ cells using a FACSCalibur (Beckton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo 6.4.7 software (Tree Star, San Carlos, CA, USA). The fluorescein isothiocyanate (FITC) channel was used to detect autofluorescence. Non-stained and non-treated cells were used as negative controls. For cell sorting experiments, cells were sorted on the basis of CD34 expression (range between 4–20%) with anti-CD34-PE on a Sony SH800Z cell sorter (Sony Biotechnology, Tokyo, Japan). CD34⁻ cells were seeded overnight. After 24 h, CD34⁻ cells were siRNA transfected using the forward transfection method (according to manufacturer’s protocol) and CD34 expression was measured on a FACSCalibur (Beckton Dickinson) at 72 h after siRNA transfection.

Cell suspensions were obtained after treatment of adherent HUVEC monolayers with TrypLE (Gibco). Cells were fixed in 2% paraformaldehyde in PBS for 15 min at room temperature and incubated with anti-CD34-phycoerythrin antibody (anti-CD34-PE; clone QBend-10, Thermo Fischer Scientific, Waltham, MA, USA), without permeabilization of the cells, to detect CD34⁺ tip cells. Cells were analyzed using a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA) and FlowJo 6.4.7 software (Tree Star, San Carlos, CA, USA). Non-stained, non-treated cells were used as negative controls. For each experiment, HUVECs of different donors were used, all with various percentages of CD34⁺ cells (~3–30%). Cell proliferation was assessed by measuring incorporation of 5-ethynyl-2’-deoxyuridine (EdU) and propidium iodide (PI) using flow cytometry, following the manufacturer’s instructions (Molecular Probes, Eugene, OR, USA).