Simultaneous Analysis of Multiple Lumenal Parameters of Individual Phagosomes Using High-Content Imaging
Simultaneous Analysis of Multiple Lumenal Parameters of Individual Phagosomes Using High-Content Imaging
复制标题
DOI：
10.1007/978-1-4939-6581-6_15
复制
发表时间：
2017-01-01
期刊：
影响因子：
--
通讯作者：
Yates, Robin M.
中科院分区：
文献类型：
--
作者：
Cheung, Samuel;Greene, Catherine;Yates, Robin M.
巨噬细胞和树突状细胞吞噬体内的生化过程对于免疫和体内平衡至关重要。吞噬体腔的可测量特性包括：评估各种水解活性、还原和氧化事件、pH、离子浓度和电化学梯度。这些通常相互依赖的吞噬体特征通常是单独评估的，这阻碍了对同一吞噬体内这些因素之间的生化关系的分析。此外，同一细胞内吞噬体自主行为的能力变得越来越明显，因此突出需要一种能够在单个吞噬体水平上对吞噬体腔化学进行多重分析的技术。在本章中，我们概述了一种方法，该方法能够利用专门设计的具有多种报告荧光的实验颗粒，结合通过自动显微镜进行的实时荧光测量，同时测量单个吞噬体的多个吞噬体参数。使用高内涵图像分析软件进行的后续分析能够以定量或半定量的方式评估每个吞噬体参数。这种方法有助于在单个吞噬体水平上实时研究巨噬细胞群中吞噬体特性之间的复杂关系。
The biochemical processes within the phagosomes of macrophages and dendritic cells are essential to immunity and homeostasis. Measurable properties of the phagosomal lumen include: assessment of various hydrolytic activities, reduction and oxidation events, pH, ion concentrations, and electrochemical gradients. These often-interdependent phagosomal features are commonly evaluated individually, hindering the analysis of the biochemical relationship between these factors within the same phagosome. In addition, the ability of phagosomes within the same cell to behave autonomously is becoming more evident, thus highlighting the need for a technique capable of multiplex analyses of phagosomal lumenal chemistries at the single phagosome level. In this chapter, we outline an approach that is capable of simultaneously measuring multiple phagosomal parameters of individual phagosomes by utilizing specifically designed experimental particles with multiple reporter fluors, in combination with real-time fluorometric measurement via automated microscopy. Subsequent analysis using high-content image analysis software enables each phagosomal parameter to be evaluated in a quantitative or semi-quantitative manner. This approach facilitates investigation of the complex relationship between phagosomal properties in a population of macrophages in real time, at the level of individual phagosomes.