Figure 3
Figure 3. VEGFR-3 is expressed in the murine BM on early megakaryocytic progenitor cells through to the megakaryoblast stage. (A) VEGFR-3 is expressed on primary murine BM cells. Murine BM cells were isolated, treated with Fc-block, and stained with Abs specific for VEGFR-3 (left panel) or an appropriate isotype control (right panel). FACS analysis showed that 1.85% ± 0.31% SEM (n = 9) of the murine BM cells expressed VEGFR-3. Dot plots of 1 representative experiment are depicted. Density plots were used to define a region in which 95% (the 2 outer contours) of the negative control events were excluded. The region was then applied to a plot displaying the stained sample. The number of positive events in both the negative control and the actual sample was then assessed. The percentage of true positive cells was calculated by subtraction of the number of events in the negative control within the defined region from the number of events found in the same region for the actual sample. Identical numbers of events were acquired. (B) VEGFR-3 is expressed on isolated mononuclear cells in the murine BM. Sections of murine femurs were stained with VEGFR-3–specific Abs (left panel, VEGFR-3; right panel, control). MK indicates megakaryocyte. Scale bars indicate 100 μm. (C) Ploidy of VEGFR-3+ cells in the murine BM. VEGFR-3+ BM cells were enriched by MACS and then analyzed in FACS. As a control, cells were treated with an appropriate isotype control. Clumping cells mimicking polyploidy were excluded from the analysis by appropriate gating strategies. The resulting histogram plot shows the DNA content of VEGFR-3+ cells. Dot plots of the DNA content of the cells were used for the quantification of VEGFR-3+ and isotype-treated cells within different ploidy classes or cell cycle stages, respectively (a detailed scheme of the gating strategy is provided in supplemental Figure 2). Subtraction of the background signal generated by unspecific binding of the isotype control allows the calculation of an actual ploidy distribution within the VEGFR-3+ population. (D) VEGFR-3 depletion has no influence on the recovery kinetics of thrombocytes and erythrocytes in lethally irradiated mice. Mice were irradiated with lethal doses of 9 Gy. Within 24 hours after irradiation, the animals were transplanted with VEGFR-3 MACS-depleted BM or complete BM as a control. Blood counts were performed regularly and the recovery of thrombocytes (left panel) and erythrocytes as a control (right panel) were monitored. (n = 5; error bars represent SEM).

VEGFR-3 is expressed in the murine BM on early megakaryocytic progenitor cells through to the megakaryoblast stage. (A) VEGFR-3 is expressed on primary murine BM cells. Murine BM cells were isolated, treated with Fc-block, and stained with Abs specific for VEGFR-3 (left panel) or an appropriate isotype control (right panel). FACS analysis showed that 1.85% ± 0.31% SEM (n = 9) of the murine BM cells expressed VEGFR-3. Dot plots of 1 representative experiment are depicted. Density plots were used to define a region in which 95% (the 2 outer contours) of the negative control events were excluded. The region was then applied to a plot displaying the stained sample. The number of positive events in both the negative control and the actual sample was then assessed. The percentage of true positive cells was calculated by subtraction of the number of events in the negative control within the defined region from the number of events found in the same region for the actual sample. Identical numbers of events were acquired. (B) VEGFR-3 is expressed on isolated mononuclear cells in the murine BM. Sections of murine femurs were stained with VEGFR-3–specific Abs (left panel, VEGFR-3; right panel, control). MK indicates megakaryocyte. Scale bars indicate 100 μm. (C) Ploidy of VEGFR-3+ cells in the murine BM. VEGFR-3+ BM cells were enriched by MACS and then analyzed in FACS. As a control, cells were treated with an appropriate isotype control. Clumping cells mimicking polyploidy were excluded from the analysis by appropriate gating strategies. The resulting histogram plot shows the DNA content of VEGFR-3+ cells. Dot plots of the DNA content of the cells were used for the quantification of VEGFR-3+ and isotype-treated cells within different ploidy classes or cell cycle stages, respectively (a detailed scheme of the gating strategy is provided in supplemental Figure 2). Subtraction of the background signal generated by unspecific binding of the isotype control allows the calculation of an actual ploidy distribution within the VEGFR-3+ population. (D) VEGFR-3 depletion has no influence on the recovery kinetics of thrombocytes and erythrocytes in lethally irradiated mice. Mice were irradiated with lethal doses of 9 Gy. Within 24 hours after irradiation, the animals were transplanted with VEGFR-3 MACS-depleted BM or complete BM as a control. Blood counts were performed regularly and the recovery of thrombocytes (left panel) and erythrocytes as a control (right panel) were monitored. (n = 5; error bars represent SEM).

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