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Nature:补充数据-通过慢性延时 2P-IVM 进行巨核细胞谱系追踪

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2024/8/17 9:34:02

Megakaryocytic lineage tracing by chronic time-lapse 2P-IVM

Nature:补充数据-通过慢性延时 2P-IVM 进行巨核细胞谱系追踪

a, Schematic showing experimental setup of chronic 2-photon intravital microscopy (2P-IVM) of mouse calvarian bone marrow. Lower: Time series of BM in VwfeGFP/+ mice. MK lineage (VWF-eGFP; green); Bone (second harmonic generation). Arrow: Fragmentation of MK results in the release of platelet-like particles. Note that after fragmentation, platelet-like particles exit the BM and new MKs grow in size before undergoing fragmentation.



Nature:补充数据-通过慢性延时 2P-IVM 进行巨核细胞谱系追踪

d, Schematic showing experimental setup of PD. Peripheral blood platelet counts monitored at indicated time points after a single injection (i.p.) of R300 or isotype control; R300: before (n = 9), 30 min (n = 3), 0.5d (n = 6), 1d (n = 8), 2d (n = 9), 4d (n = 9), 8d (n = 4) mice; isotype: before (n = 7), 30 min (n = 3), 0.5d (n = 4), 1d (n = 5), 2d (n = 7), 4d (n = 4), 8d (n = 3) Mean ± SD.e, Morphometric analysis of MKs shows decreased sphericity in response to PD corresponding to an increase of cellular protrusions; Control n = 63 cells and PD 12 h n = 51 cells, pooled from 4 mice; unpaired t-test/Welch’ correction, ***: p = 0.0002; Mean ± SD. Scale bars = 50 μm. f, Representative 2P-IVM time series of proplatelet formation and MK fragmentation. Single cell tracking of MK volumes over time reveals a significant faster decrease of volume following PD. Control n = 14 and PD n = 11 cells, pooled from 3 mice; unpaired t-test; ns: p = 0.0206; Mean ± SD. g, Upper: Small ( < 15 μm) VWF-eGFP+ cells (arrows) appear 12 h after PD (2P-IVM). Also see Fig. 1i. Scale bar = 50 µm. Middle: PD triggers an instantaneous proliferation of MKPs peaking 1d following platelet depletion; MKPs (CD41+/CD42–) were counted in whole-mount BMs. n = 3 mice; one-way ANOVA/Dunnett, **: p = 0.0023, ****: p = 0.000009, ***: p = 0.0008, ns: p = 0.063 and p = 0.072; Mean ± SD. Lower: Proliferation of MKPs (VWF-eGFP+/CD41+/CD42–) was measured after in vivo labelling of BM cells with 5-ethynyl-2′-deoxyuridine (EdU) using FACS. Mean fluorescent intensity of EdU and Frequency of EdU-pve cells significantly increases after PD (12 h); n = 3 mice; paired t-test; **: p = 0.0046; *: p = 0.0115; error bar=SD; Mean ± SD. h, Left: MKPs lodged within the perivascular niche of the BM grow in volume. Volume increase of single cells was tracked over time. Arrested MKP n = 27 cells and mobile MKP n = 6 cells. Notably, the speed of cell growth after PD did not significantly differ from steady state control; Control n = 54 cells and PD = 18 cells, pooled from 3 mice, unpaired t-test Welch’s correction ns: p = 0.7503; Mean ± SD.


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Nature:补充数据-通过慢性延时 2P-IVM 进行巨核细胞谱系追踪


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