Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel.
Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel.
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DOI：
10.1039/b719799c
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发表时间：
2008-10
期刊：
影响因子：
6.1
通讯作者：
Wikswo JP
中科院分区：
文献类型：
--
作者：
Faley S;Seale K;Hughey J;Schaffer DK;VanCompernolle S;McKinney B;Baudenbacher F;Unutmaz D;Wikswo JP
解读抗原呈递细胞通过免疫突触的形成调控naïve CD4+ T辅助细胞刺激的信号通路是基本理解成功的适应性免疫反应进展的关键。然而，体外T细胞- APC相互作用的研究是具有挑战性的，因为随着时间的推移难以跟踪单个非贴壁细胞对。随着时间的推移，研究单细胞动力学揭示了可能在批量实验中平均的罕见但关键的信号事件，但这些不太常见的事件无疑对细胞对其微环境的反应的综合理解是重要的。我们描述了一种微流体技术的新应用，它克服了传统细胞培养的许多限制，使数百个被动隔离的造血细胞能够长时间研究。该微流体细胞陷阱装置由440个18 μm×18 μm×10 μm的PDMS组成，桶状结构与流动方向相反，当细胞通过细胞陷阱区域时，桶状结构可以作为细胞的畜栏。细胞活力分析显示，超过70%的naïve CD4+ T细胞（TN）仅使用水动力保持在一定位置，随后可存活24小时。在引入化学、抗体或细胞形式的刺激后，TN细胞成功诱导胞质钙瞬变。单次刺激实验对TN细胞的统计分析揭示了该平台区分不同钙反应模式的能力，这种能力可能被用来表征特定人群中T细胞信号传导状态。最后，我们研究了原代T细胞和树突状细胞之间的实时接触和非接触相互作用，这是免疫突触形成的两个主要参与者。利用菊花链结构中的微流体陷阱，我们可以观察到仅暴露于脂多糖成熟树突状细胞分泌物培养基中的TN细胞中的钙瞬态，这一事件在传统细胞培养中很容易被遗漏，因为大的培养基与细胞比稀释了细胞产物。对这一细胞间信号事件的进一步研究表明，lps成熟的树突状细胞在缺乏抗原刺激的情况下，会分泌化学信号诱导TN细胞钙瞬变。虽然成熟树突状细胞产生的刺激因子仍有待确定，但本报告说明了这些微流体细胞陷阱在分析单个悬浮细胞随时间排列和探测一个或多个细胞群体之间基于接触和细胞间信号事件方面的效用。
Deciphering the signaling pathways that govern stimulation of naïve CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell – APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, nonadherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 μm×18 μm×10 μm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of naïve CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real-time contact and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in TN cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and inter-cellular signaling events between one or more cell populations.
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DOI：
10.1084/jem.184.2.777
发表时间：
1996-08-01
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Beeson C;Rabinowitz J;Tate K;Gütgemann I;Chien YH;Jones PP;Davis MM;McConnell HM
通讯作者：
McConnell HM
影响因子：
4.5
作者：
Eklund, SE;Snider, RM;Cliffel, DE
通讯作者：
Cliffel, DE
影响因子：
5.4
作者：
Pajusto, M;Ihalainen, N;Mattila, PS
通讯作者：
Mattila, PS
影响因子：
6.1
作者：
Di Carlo, Dino;Wu, Liz Y.;Lee, Luke P.
通讯作者：
Lee, Luke P.
DOI：
10.1124/jpet.106.108209
发表时间：
2006-10-01
影响因子：
3.5
作者：
Navarrete, Carmen;Sancho, Rocio;Munoz, Eduardo
通讯作者：
Munoz, Eduardo