Suvannapruk Waraporn, Edney Max K, Fisher Leanne E, Luckett Jeni C, Kim Dong-Hyun, Scurr David J, Ghaemmaghami Amir M, Alexander Morgan R
Advanced Materials and Healthcare Technologies Division, School of Pharmacy, University of Nottingham, University Park Nottingham, NG7 2RD, UK.
Department of Chemical and Environmental Engineering, Faculty of Engineering, University of Nottingham, University Park Nottingham, NG7 2RD, UK.
Bio Protoc. 2023 Aug 5;13(15):e4727. doi: 10.21769/BioProtoc.4727.
The Three-dimensional OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) is a secondary ion mass spectrometry instrument, a combination of a Time of Flight (ToF) instrument with an Orbitrap analyzer. The 3D OrbiSIMS technique is a powerful tool for metabolic profiling in biological samples. This can be achieved at subcellular spatial resolution, high sensitivity, and high mass-resolving power coupled with MS/MS analysis. Characterizing the metabolic signature of macrophage subsets within tissue sections offers great potential to understand the response of the human immune system to implanted biomaterials. Here, we describe a protocol for direct analysis of individual cells after in vitro differentiation of naïve monocytes into M1 and M2 phenotypes using cytokines. As a first step in vivo, we investigate explanted silicon catheter sections as a medical device in a rodent model of foreign body response. Protocols are presented to allow the host response to different immune instructive materials to be compared. The first demonstration of this capability illustrates the great potential of direct cell and tissue section analysis for in situ metabolite profiling to probe functional phenotypes using molecular signatures. Details of the in vitro cell approach, materials, sample preparation, and explant handling are presented, in addition to the data acquisition approaches and the data analysis pipelines required to achieve useful interpretation of these complex spectra. This method is useful for in situ characterization of both in vitro single cells and ex vivo tissue sections. This will aid the understanding of the immune response to medical implants by informing the design of immune-instructive biomaterials with positive interactions. It can also be used to investigate a broad range of other clinically relevant therapeutics and immune dysregulations. Graphical overview.
三维轨道阱二次离子质谱仪（3D OrbiSIMS）是一种二次离子质谱仪，它是飞行时间（ToF）仪器与轨道阱分析仪的组合。3D OrbiSIMS技术是用于生物样品代谢谱分析的强大工具。这可以在亚细胞空间分辨率、高灵敏度和高质量分辨能力以及串联质谱分析的条件下实现。表征组织切片内巨噬细胞亚群的代谢特征为理解人类免疫系统对植入生物材料的反应提供了巨大潜力。在这里，我们描述了一种在体外使用细胞因子将幼稚单核细胞分化为M1和M2表型后直接分析单个细胞的方案。作为体内的第一步，我们在异物反应的啮齿动物模型中研究作为医疗器械的植入硅导管切片。还提供了相关方案，以便比较宿主对不同免疫诱导材料的反应。这种能力的首次展示说明了直接细胞和组织切片分析在原位代谢物谱分析中利用分子特征探测功能表型的巨大潜力。除了实现对这些复杂光谱进行有用解释所需的数据采集方法和数据分析流程外，还介绍了体外细胞方法、材料、样品制备和外植体处理的详细信息。该方法对于体外单细胞和离体组织切片的原位表征很有用。这将有助于通过设计具有积极相互作用的免疫诱导生物材料来理解对医疗植入物的免疫反应。它还可用于研究广泛的其他临床相关治疗方法和免疫失调。图形概述。
