Fitc conjugated anti annexin 5

Apoptotic cell death in CD34⁻ and CD34⁺ cells were determined at 72 h after siRNA transfection. Cells were stained using FITC-conjugated anti-annexin V (Life technologies, Eugene, OR, USA) and anti-CD34-PE (Thermo Scientific), according to the manufacturer’s instructions. The apoptotic fraction of CD34⁻ and CD34⁺ cells was detected by flow cytometry using a FACSCalibur (Beckton Dickinson) and analyzed using FlowJo 6.4.7 software (Tree Star). As a control for the role of glucose oxidation in tip cell survival, cells were treated with UK5099 (2 µM; Sigma-Aldrich), a compound that blocks the channeling of pyruvate into mitochondrial respiration, for 24 h in HUVEC medium and the percentage of tip cells was measured as described above.

Apoptotic cell death in CD34^- and CD34⁺ cells was determined at 72 h after siRNA transfection. Cells were stained using FITC-conjugated anti-annexin V (Life Technologies, Eugene, OR, USA) and anti-CD34-PE (Thermo Scientific), according to the manufacturer’s instructions. The apoptotic fraction of CD34^- and CD34⁺ cells was detected by flow cytometry using a FACSCalibur (Beckton Dickinson) and analyzed using FlowJo 6.4.7 software (Tree Star).