Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap
Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap
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DOI：
10.1038/s41596-020-0354-0
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发表时间：
2020-08-12
期刊：
影响因子：
14.8
通讯作者：
Vedrine, Christophe
中科院分区：
文献类型：
--
作者：
Bounab, Yacine;Eyer, Klaus;Vedrine, Christophe
目前，缺乏能够对单个细胞进行定量和动态表型表征的系统，特别是对细胞因子和抗体等分泌蛋白质的分析，阻碍了免疫反应的表征。我们最近开发了一个简单而强大的微流控平台DropMap，用于同时测量数万个单个免疫细胞的分泌动力学和其他细胞特征，包括内吞活性、活力和细胞表面标志的表达。单个细胞被分隔在50-Pl液滴中，并使用荧光显微镜结合基于荧光重新定位到顺磁性纳米颗粒的免疫分析进行分析，顺磁性纳米颗粒在磁场中排列形成珠线。该方案通常在微流控芯片和室的准备之后花费8-10小时，这可以提前完成。相比之下，酶联免疫斑点(ELISPOT)、流式细胞仪、飞行时间质谱仪(CyTOF)和单细胞测序只能实现终点测量，而不能直接、定量地测量分泌蛋白。我们说明了该系统如何用于描述感染性休克患者单个单核细胞分泌肿瘤坏死因子-α的下调，通过测量单个T细胞分泌细胞因子的速率来研究免疫反应，以及测量单个B细胞分泌的抗体的亲和力。该协议描述了一个用于动态高通量分析单个细胞表型的微流体平台。细胞表面标志物和分泌的蛋白质被量化，并通过定制免疫分析的荧光检测来表征。
Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8-10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-alpha (TNF-alpha) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.This protocol describes a microfluidic platform for dynamic high-throughput analysis of the phenotypes of single cells. Cell-surface markers and secreted proteins are quantified and characterized by fluorescence detection using tailored immunoassays.