Figure 2.
In situ fluorescence labeling, light sheet fluoresence microscopy (LSFM), 2-photon intravital microscopy (2PIVM), and ex vivo proplatelet assays demonstrate that the bone marrow is the primary site of megakaryopoiesis in adult mice. (A) Micrographs of cryosections obtained from mouse fetal liver, bone marrow, spleen, and lung, respectively. Blue, 4′,6-diamidino-2-phenylindole (DAPI); cyan, glycoprotein IX (GPIX); yellow, platelet factor 4 (PF4); and magenta, laminin. Scale bars represent 50 μm. (B) Quantitative analysis of MK count within whole organ cryosections (PF4) MK/mm2; blue, fetal liver; red, bone marrow; green, spleen; and light blue, lung. (C) Reconstruction of LSFM data of femoral bone marrow, spleen, and lung tissues. MKs are depicted in cyan (anti-GPIX) and vessels in magenta (anti-CD105); scale bars represent 100 μm. (D) Quantification of MK (GPIX+ > 16 μm) numbers per mm³ in the adult mouse bone marrow, spleen, and lung; bar graphs represent mean ± standard deviation. Red, bone marrow; green, spleen; and light blue, lung. (E) Representative images of 2PIVM data of adult mouse bone marrow, spleen, and lung tissues. MKs (eGFP+ > 16 μm) are depicted in cyan and vessels in magenta (Evans blue; scale bars represent 50 μm). (F) Quantification of 2PIVM data shows an average number of MKs per mouse. Red, bone marrow; green, spleen; and light blue, lung. (G) Representative brightfield images of day 4 mouse fetal liver, bone marrow, spleen, and lung cultures pregradient. White arrows indicate MKs. Scale bars represent 50 μm. (H) Representative micrographs of proplatelet-forming MKs differentiated ex vivo from mouse fetal liver, bone marrow, and spleen progenitors. Blue, DAPI; magenta, CD41+; cyan, α-tubulin. Scale bars represent 10 μm. (I) Quantitative analysis of MK counts on day 4 MKs differentiated ex vivo; blue, fetal liver; red, bone marrow; green, spleen; and light blue, lung. (J) Representative phase contrast images of proplatelet-forming MKs from multiple tissues at 24 hours time points. Red, MKs; green, proplatelet extensions. Scale bars represent 100 μm. (K) Quantitative analysis of the percentage of MKs forming proplatelets; blue, fetal liver; red, bone marrow; and green, spleen. (L) Total proplatelet area of MKs forming proplatelets; blue, fetal liver; red, bone marrow; and green, spleen. (M) Percentage of MKs forming proplatelets in shear conditions. Blue, fetal liver; red, bone marrow; and green, spleen. (N) Representative panels of shear-driven proplatelet formation and extension in a microfluidic device over time for ex vivo differentiated MKs from multiple tissues. The time scale is T = 0 minute → 4 minutes. Scale bars represent 50 μm. Unless otherwise stated, all data sets are shown as mean ± standard error of the mean; graphs represent data from a minimum of 3 independent mice. Statistics were performed with 1-way analysis of variance with Tukey multiple comparisons test at 95% CI.

In situ fluorescence labeling, light sheet fluoresence microscopy (LSFM), 2-photon intravital microscopy (2PIVM), and ex vivo proplatelet assays demonstrate that the bone marrow is the primary site of megakaryopoiesis in adult mice. (A) Micrographs of cryosections obtained from mouse fetal liver, bone marrow, spleen, and lung, respectively. Blue, 4′,6-diamidino-2-phenylindole (DAPI); cyan, glycoprotein IX (GPIX); yellow, platelet factor 4 (PF4); and magenta, laminin. Scale bars represent 50 μm. (B) Quantitative analysis of MK count within whole organ cryosections (PF4) MK/mm2; blue, fetal liver; red, bone marrow; green, spleen; and light blue, lung. (C) Reconstruction of LSFM data of femoral bone marrow, spleen, and lung tissues. MKs are depicted in cyan (anti-GPIX) and vessels in magenta (anti-CD105); scale bars represent 100 μm. (D) Quantification of MK (GPIX+ > 16 μm) numbers per mm³ in the adult mouse bone marrow, spleen, and lung; bar graphs represent mean ± standard deviation. Red, bone marrow; green, spleen; and light blue, lung. (E) Representative images of 2PIVM data of adult mouse bone marrow, spleen, and lung tissues. MKs (eGFP+ > 16 μm) are depicted in cyan and vessels in magenta (Evans blue; scale bars represent 50 μm). (F) Quantification of 2PIVM data shows an average number of MKs per mouse. Red, bone marrow; green, spleen; and light blue, lung. (G) Representative brightfield images of day 4 mouse fetal liver, bone marrow, spleen, and lung cultures pregradient. White arrows indicate MKs. Scale bars represent 50 μm. (H) Representative micrographs of proplatelet-forming MKs differentiated ex vivo from mouse fetal liver, bone marrow, and spleen progenitors. Blue, DAPI; magenta, CD41+; cyan, α-tubulin. Scale bars represent 10 μm. (I) Quantitative analysis of MK counts on day 4 MKs differentiated ex vivo; blue, fetal liver; red, bone marrow; green, spleen; and light blue, lung. (J) Representative phase contrast images of proplatelet-forming MKs from multiple tissues at 24 hours time points. Red, MKs; green, proplatelet extensions. Scale bars represent 100 μm. (K) Quantitative analysis of the percentage of MKs forming proplatelets; blue, fetal liver; red, bone marrow; and green, spleen. (L) Total proplatelet area of MKs forming proplatelets; blue, fetal liver; red, bone marrow; and green, spleen. (M) Percentage of MKs forming proplatelets in shear conditions. Blue, fetal liver; red, bone marrow; and green, spleen. (N) Representative panels of shear-driven proplatelet formation and extension in a microfluidic device over time for ex vivo differentiated MKs from multiple tissues. The time scale is T = 0 minute → 4 minutes. Scale bars represent 50 μm. Unless otherwise stated, all data sets are shown as mean ± standard error of the mean; graphs represent data from a minimum of 3 independent mice. Statistics were performed with 1-way analysis of variance with Tukey multiple comparisons test at 95% CI.

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