Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform.
Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform.
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使用多参数光维元平台对钙流入和HIV-1感染的单细胞表征。
DOI：
10.3791/62632
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发表时间：
2021-05-18
影响因子：
1.2
通讯作者：
Swartz, Talia H.
中科院分区：
文献类型：
--
作者：
Freeman, Tracey;Andreou, Christina;Sebra, Robert P.;Beaumont, Kristin G.;Swartz, Talia H.
HIV-1是一种慢性感染，影响着全世界3700多万人。人类免疫缺陷病毒（HIV）感染者尽管接受了抗逆转录病毒治疗，但仍会经历与慢性炎症相关的合并症。然而，这些炎症信号尚未得到充分表征。早期进入事件对细胞信号传导事件和下游基因表达的激活的作用尚未在单细胞水平上被捕获。在这里，作者描述了一种方法，该方法将活细胞荧光显微镜的原理应用于自动化单细胞平台，该平台在用户自定义的时间过程中培养和成像细胞，从而实现动态细胞过程的高通量分析。该检测可以跟踪HIV-1感染后立即发生的早期事件的单细胞活荧光显微镜，特别是伴随暴露于病毒的钙的流入和使用荧光报告病毒的生产性感染的发展。MT-4细胞装载有钙敏感性染料，并在纳米流体装置上的隔离围栏中培养。用HIV-1报告病毒（HIV-1 NLCI）感染培养的细胞。位于纳米流体装置上方的荧光显微镜测量急性HIV-1暴露后8分钟内的钙流入。HIV-1生产性感染在这些相同的细胞中测量4天的间隔。分析来自这些时间过程的成像数据以定义病毒-宿主受体相互作用和信号传导途径动力学。作者提出了一种集成的、可扩展的替代传统成像方法的方法，该方法使用一种新型的光流体平台，能够进行单细胞分选、培养、成像和软件自动化。该测定可测量各种条件下的事件动力学，包括细胞类型、激动剂或拮抗剂效应，同时测量一系列参数。这是第一个建立的用于纳米流体高通量纵向单细胞培养和成像的方法：该技术可以广泛适用于研究细胞信号动力学和动态分子相互作用。
HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.
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影响因子：
7.3
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通讯作者：
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