A 3D Tissue-wide Digital Imaging Pipeline for Quantitation of Secreted Molecules Shows Absence of CXCL12 Gradients in Bone Marrow
A 3D Tissue-wide Digital Imaging Pipeline for Quantitation of Secreted Molecules Shows Absence of CXCL12 Gradients in Bone Marrow
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DOI：
10.1016/j.stem.2019.10.003
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发表时间：
2019-12-05
期刊：
影响因子：
23.9
通讯作者：
Schroeder, Timm
中科院分区：
文献类型：
--
作者：
Kunz, Leo;Schroeder, Timm
技术限制阻碍了对个体分子（包括假定的干细胞调节因子）如何分布在组织和干细胞壁龛中的理解。在这里，我们报告了使用集成的3D定量策略和软件，使用多个附加荧光通道对单个蛋白质进行大体积原位成像的近距离结联试验（PLA）的适应性。使用该平台，我们量化了单个CXCL12趋化因子蛋白在粒细胞集落刺激因子（G-CSF）处理前后的骨髓（BM）分布。我们发现无处不在的CXCL12分布具有局部富集但没有远程梯度，这与目前关于CXCL12如何控制BM内造血干细胞和祖细胞（HSPCs）迁移的假设相反。这种离散数字定量、大容量、多色成像管道具有单分子灵敏度，可广泛应用于任何抗体表位和组织，从而进一步深入了解组织的分子组织和细胞相互作用。
Technological limitations have hampered understanding of how individual molecules, including putative stem cell regulators, are distributed throughout tissues and stem cell niches. Here, we report adaptation of the proximity ligation assay (PLA) for large-volume, in situ imaging of individual proteins with multiple additional fluorescent channels with integrated 3D quantification strategies and software. Using this platform, we quantified the bone marrow (BM) distribution of individual CXCL12 chemokine proteins, both before and after their depletion by granulocyte-colony stimulating factor (G-CSF) treatment. We found ubiquitous CXCL12 distributions with local enrichments but no long-range gradients, in contrast to current assumptions about how CXCL12 controls migration of hematopoietic stem and progenitor cells (HSPCs) within BM. This pipeline for discrete digital quantitative, large-volume, multicolor imaging, with up to single-molecule sensitivity, may be broadly applied to any antibody epitope and tissue, enabling further insights into molecular organization of tissues and cellular interactions.