Mitochondrial respiration in tip cell and non-tip cell HUVECs. (a) Tip cells showed higher total ATP levels than non-tip cells (measured as relative luminescence). (b) Mitochondrial respiration in CD34- and CD34+ FACS-sorted HUVECs. (c) Quantification of OCR in non-tip cells and tip cells revealed no difference in basal mitochondrial respiration and ATP production, but tip cells showed a higher maximum capacity and spare capacity of mitochondrial respiration. (d) Mitochondrial DNA revealed no differences between non-tip cells and tip cells. (e) Blocking mitochondrial respiration with oligomycin A (1.5 µM) and antimycin A (2.5 µM)/rotenone (1.25 µM) in tip cells and non-tip cells induced a switch to glycolysis, but to a lesser extent in tip cells. Mitochondrial fuel oxidation analysis showed the dependency (f) of tip cells and non-tip cells on the use of glucose, glutamine, and FAO, and their capacity (g) to oxidize these substrates in mitochondrial respiration. Tip cells showed a higher dependency on glucose and glutamine oxidation, whereas non-tip cells showed a higher dependency on FAO. Tip cells showed a higher capacity for glutamine and glucose oxidation, whereas non-tip cells showed a higher FAO capacity. OCR measurements were represented as fold change compared to basal OCR in non-tip cells. Luminescence was normalized for cell numbers. Results are shown as means ± SEM of experiments with HUVECs of at least 3 donors. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to non-tip cells (Unpaired Student’s t-test).