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Electrophoretic cytometry of adherent cells
Lab on a Chip ( IF 6.1 ) Pub Date : 2017-11-06 00:00:00 , DOI: 10.1039/c7lc01012e
Elaine J. Su 1, 2, 3, 4, 5 , Amy E. Herr 1, 2, 3, 4
Affiliation  

Cell–matrix and cell–cell interactions influence intracellular signalling and play an important role in physiologic and pathologic processes. Detachment of cells from the surrounding microenvironment alters intracellular signalling. Here, we demonstrate and characterise an integrated microfluidic device to culture single and clustered cells in tuneable microenvironments and then directly analyse the lysate of each cell in situ, thereby eliminating the need to detach cells prior to analysis. First, we utilise microcontact printing to pattern cells in confined geometries. We then utilise a microscale isoelectric focusing (IEF) module to separate, detect, and analyse lamin A/C from substrate-adhered cells seeded and cultured at varying (500, 2000, and 9000 cells per cm2) densities. We report separation performance (minimum resolvable pI difference of 0.11) that is on par with capillary IEF and independent of cell density. Moreover, we map lamin A/C and β-tubulin protein expression to morphometric information (cell area, circumference, eccentricity, form factor, and cell area factor) of single cells and observe poor correlation with each of these parameters. By eliminating the need for cell detachment from substrates, we enhance detection of cell receptor proteins (CD44 and β-integrin) and dynamic phosphorylation events (pMLCS19) that are rendered undetectable or disrupted by enzymatic treatments. Finally, we optimise protein solubilisation and separation performance by tuning lysis and electrofocusing (EF) durations. We observe enhanced separation performance (decreased peak width) with longer EF durations by 25.1% and improved protein solubilisation with longer lysis durations. Overall, the combination of morphometric analyses of substrate-adhered cells, with minimised handling, will yield important insights into our understanding of adhesion-mediated signalling processes.

中文翻译:

贴壁细胞电泳细胞仪

细胞基质和细胞间的相互作用影响细胞内信号传导,并在生理和病理过程中发挥重要作用。从周围微环境中分离细胞会改变细胞内信号传导。在这里,我们演示并表征了集成的微流控设备,可在可调节的微环境中培养单个和成簇的细胞,然后直接在原位分析每个细胞的裂解物,从而消除了在分析之前分离细胞的需要首先,我们利用微接触印刷在受限的几何形状中对单元进行图案化。然后,我们利用微型等电聚焦(IEF)模块从以每平方厘米2(500、2000和9000个细胞)播种和培养的基质粘附细胞中分离,检测和分析板层蛋白A / C。)的密度。我们报告的分离性能(最小可分辨pI差异为0.11)与毛细管IEF相同,并且与细胞密度无关。此外,我们将lamin A / C和β-微管蛋白蛋白表达映射到单个细胞的形态计量信息(细胞面积,周长,离心率,形状因子和细胞面积因子),并观察到与这些参数中的每一个之间的相关性很差。通过消除细胞与底物分离的需要,我们增强了细胞受体蛋白(CD44和β-整合素)的检测以及动态磷酸化事件(pMLC S19),使其无法被酶处理或破坏。最后,我们通过调整裂解和电聚焦(EF)的持续时间来优化蛋白质的溶解和分离性能。我们观察到更长的EF持续时间可提高25.1%的分离性能(峰宽减小),而更长的裂解时间可改善蛋白质的溶解度。总体而言,结合底物粘附细胞的形态分析与最小限度的处理将为我们对粘附介导的信号传导过程的理解提供重要的见识。
更新日期:2017-11-09
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