Ng Alphonsus H C, Chamberlain M Dean, Situ Haozhong, Lee Victor, Wheeler Aaron R
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S 3G9, Canada.
Donnelly Centre for Cellular and Biomolecular Research, 160 College Street, Toronto, Ontario M5S 3E1, Canada.
Nat Commun. 2015 Jun 24;6:7513. doi: 10.1038/ncomms8513.
We report a new technique called Digital microfluidic Immunocytochemistry in Single Cells (DISC). DISC automates protocols for cell culture, stimulation and immunocytochemistry, enabling the interrogation of protein phosphorylation on pulsing with stimulus for as little as 3 s. DISC was used to probe the phosphorylation states of platelet-derived growth factor receptor (PDGFR) and the downstream signalling protein, Akt, to evaluate concentration- and time-dependent effects of stimulation. The high time resolution of the technique allowed for surprising new observations-for example, a 10 s pulse stimulus of a low concentration of PDGF is sufficient to cause >30% of adherent fibroblasts to commit to Akt activation. With the ability to quantitatively probe signalling events with high time resolution at the single-cell level, we propose that DISC may be an important new technique for a wide range of applications, especially for screening signalling responses of a heterogeneous cell population.
我们报告了一种名为单细胞数字微流控免疫细胞化学（DISC）的新技术。DISC可实现细胞培养、刺激和免疫细胞化学协议的自动化，能够在仅3秒的刺激脉冲下检测蛋白质磷酸化情况。DISC用于探测血小板衍生生长因子受体（PDGFR）和下游信号蛋白Akt的磷酸化状态，以评估刺激的浓度和时间依赖性效应。该技术的高时间分辨率带来了令人惊讶的新发现——例如，低浓度血小板衍生生长因子（PDGF）的10秒脉冲刺激足以使>30%的贴壁成纤维细胞激活Akt。由于能够在单细胞水平以高时间分辨率定量探测信号事件，我们认为DISC可能是一种适用于广泛应用的重要新技术，特别是用于筛选异质细胞群体的信号反应。
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