Fig. 2: Quantification of platelets generated by passage of megakaryocytes through mouse pulmonary vasculature ex vivo.

Mouse megakaryocytes (MKs), labelled with CD41-PE or CD41-FITC antibodies, were passaged repeatedly through the pulmonary vasculature ex vivo. Lungs were ventilated with air, pure nitrogen or without ventilation. a The number of generated platelets per MK present in the perfusates from different passage numbers in lungs either ventilated with air (black circles), pure nitrogen (red triangles), or without ventilation (blue squares) were measured by FACS. N = 5 (air-ventilated lung or unventilated lung) and 4 (nitrogen-ventilated lung) independent experiments. Data are mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. b Stained MKs (CD41-FITC, green) were passaged through pulmonary vasculature ex vivo 18 times, and lung tissue was fixed and sliced followed by visualization of 20 stacked focal planes by two-photon microscopy. Lungs were either ventilated with air or pure nitrogen, or were not ventilated, as indicated. Mouse lung without MKs passed through served as control. Images shown are representative of n ≥ 4 independent experiments. scale bar: 20 µm. c Numbers of platelets generated per MK in perfusate and retained in mouse lung under air ventilation, were calculated and displayed as mean ± SEM. As a control, MKs passed through 21 G needles 18 times generated no platelets. N = 6 (air-ventilated lung) and 4 (passing through needles) independent experiments. Source data are provided in the Source Data file.