Nuclear translocation kinetics of NF-κB in macrophages challenged with pathogens in a microfluidic platform
Nuclear translocation kinetics of NF-κB in macrophages challenged with pathogens in a microfluidic platform
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DOI：
10.1007/s10544-008-9281-5
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发表时间：
2009-06-01
影响因子：
2.8
通讯作者：
Singh, Anup K.
中科院分区：
文献类型：
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作者：
James, Conrad D.;Moorman, Matthew W.;Singh, Anup K.
我们已经开发了一个微流控平台，用于实时成像宿主-病原体相互作用和细胞信号传导事件。将宿主细胞固定在受控环境中，用于光学询问对病原体挑战的免疫应答的动力学和随机性。在这里，我们已经定量测量了在RAW264.7小鼠巨噬细胞样细胞中Toll样受体4（TLR 4）通路的激活。这是通过测量绿色荧光蛋白融合构建体与NF-κ B转录因子亚基RelA（GFP-RelA）的胞质至核易位动力学来实现的。测量了响应活细菌和纯化脂多糖（LPS）挑战的易位动力学，并且这项工作首次展示了在微流体平台上对宿主细胞感染的实时成像，并对早期（感染后< 0.5小时）免疫信号传导事件进行定量分析。我们的数据显示，LPS剂量增加1,000倍导致我们开发的宿主细胞活化度量增加10倍，以描述NF-κ B易位动力学。使用该度量，活细菌激发被分配等效的LPS剂量，作为比较仅TLR 4途径信号传导（由LPS激活）和多途径信号传导（由全细菌激活）之间的NF-κ B易位动力学的第一步。该装置还包含用于捕获和流体分离单个宿主细胞的独特架构，以区分原发性和继发性免疫信号传导。
We have developed a microfluidic platform for real-time imaging of host-pathogen interactions and cellular signaling events. Host cells are immobilized in a controlled environment for optical interrogation of the kinetics and stochasticity of immune response to pathogenic challenges. Here, we have quantitatively measured activation of the toll-like receptor 4 (TLR4) pathway in RAW264.7 murine macrophage-like cells. This was achieved by measuring the cytoplasm-to-nucleus translocation kinetics of a green fluorescent protein fusion construct to the NF-kappa B transcription factor subunit RelA (GFP-RelA). Translocation kinetics in response to live bacteria and purified lipopolysaccharide (LPS) challenges were measured, and this work presents the first demonstration of live imaging of host cell infection on a microfluidic platform with quantitative analysis of an early (< 0.5 h from infection) immune signaling event. Our data show that a 1,000x increase in the LPS dose led to a similar to 10x increase in a host cell activation metric we developed in order to describe NF-kappa B translocation kinetics. Using this metric, live bacteria challenges were assigned an equivalent LPS dose as a first step towards comparing NF-kappa B translocation kinetics between TLR4-only pathway signaling (activated by LPS) and multiple pathway signaling (activated by whole bacteria). The device also contains a unique architecture for capturing and fluidically isolating single host cells for the purpose of differentiating between primary and secondary immune signaling.