Spatial Single Cell Analysis of Proteins in 2D Human Gastruloids Using Iterative Immunofluorescence.
Spatial Single Cell Analysis of Proteins in 2D Human Gastruloids Using Iterative Immunofluorescence.
复制标题
使用迭代免疫荧光对 2D 人类原肠胚中的蛋白质进行空间单细胞分析。
DOI：
10.1002/cpz1.915
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Heemskerk,Idse
中科院分区：
文献类型：
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作者：
Freeburne,Emily;Teague,Seth;Khan,Hina;Li,Bolin;Ding,Siyuan;Chen,Bohan;Helms,Adam;Heemskerk,Idse
在发育过程中，细胞信号传导指示组织模式，最初相同的细胞产生由不同细胞类型组成的空间组织结构的过程。多个信号如何在空间和时间中组合指示命运仍然知之甚少。同时测量通过多个信号传导途径的信号传导活性和它们控制的细胞命运对于解决这个问题至关重要。在这里，我们描述了一个迭代的免疫荧光协议和计算管道，以询问模式的形成在一个2D模型的人原肠胚具有更大的复用比标准的免疫荧光技术是可能的。该协议和计算管道一起实现了成像，然后对相同胃类中超过27种蛋白质进行空间和共定位分析。我们通过使用scRNA-seq熟悉的技术对单细胞蛋白表达进行聚类，并将其与空间位置联系起来，以计算不同细胞类型的空间分布和细胞信号传导活性，从而证明了这一点。这些方法不限于在2D gastruloids的图案，可以很容易地扩展到其他情况下。除了迭代免疫荧光方案和分析流程外，还包括2D胃状体分化和生产微图案化多孔载玻片的支持方案。版权所有© 2023作者。当前方案由Wiley Periodicals LLC出版。基本方案1：迭代免疫荧光基本方案2：计算分析管道支持方案1：生成微图案化多孔载玻片支持方案2：2D类胃体的分化
During development, cell signaling instructs tissue patterning, the process by which initially identical cells give rise to spatially organized structures consisting of different cell types. How multiple signals combinatorially instruct fate in space and time remains poorly understood. Simultaneous measurement of signaling activity through multiple signaling pathways and of the cell fates they control is critical to addressing this problem. Here we describe an iterative immunofluorescence protocol and computational pipeline to interrogate pattern formation in a 2D model of human gastrulation with far greater multiplexing than is possible with standard immunofluorescence techniques. This protocol and computational pipeline together enable imaging followed by spatial and co‐localization analysis of over 27 proteins in the same gastruloids. We demonstrate this by clustering single cell protein expression, using techniques familiar from scRNA‐seq, and linking this to spatial position to calculate spatial distributions and cell signaling activity of different cell types. These methods are not limited to patterning in 2D gastruloids and can be easily extended to other contexts. In addition to the iterative immunofluorescence protocol and analysis pipeline, Support Protocols for 2D gastruloid differentiation and producing micropatterned multi‐well slides are included. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Iterative immunofluorescenceBasic Protocol 2: Computational analysis pipelineSupport Protocol 1: Generating micropatterned multi‐well slidesSupport Protocol 2: Differentiation of 2D gastruloids