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Nature:補充數據-通過慢性延時 2P-IVM 進行巨核細胞譜系追蹤

更新時間:2024-08-17   點擊次數:415次

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

Nature:補充數據-通過慢性延時 2P-IVM 進行巨核細胞譜系追蹤

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:補充數據-通過慢性延時 2P-IVM 進行巨核細胞譜系追蹤

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:補充數據-通過慢性延時 2P-IVM 進行巨核細胞譜系追蹤


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