Identification and Sorting of Tip Cells

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.