Cerveira Joana, Begum Julfa, Di Marco Barros Rafael, van der Veen Annemarthe G, Filby Andrew
FACS Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, Holborn WC2A 3LY, UK.
Immuno Surveillance Laboratory London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, Holborn, London WC2A 3LY, UK.
J Immunol Methods. 2015 Aug;423:120-30. doi: 10.1016/j.jim.2015.04.030. Epub 2015 May 9.
Calcium ions (Ca(2+)) are a ubiquitous transducer of cellular signals controlling key processes such as proliferation, differentiation, secretion and metabolism. In the context of T cells, stimulation through the T cell receptor has been shown to induce the release of Ca(2+) from intracellular stores. This sudden elevation within the cytoplasm triggers the opening of ion channels in the plasma membrane allowing an influx of extracellular Ca(2+) that in turn activates key molecules such as calcineurin. This cascade ultimately results in gene transcription and changes in the cellular state. Traditional methods for measuring Ca(2+) include spectrophotometry, conventional flow cytometry (CFC) and live cell imaging techniques. While each method has strengths and weaknesses, none can offer a detailed picture of Ca(2+) mobilisation in response to various agonists. Here we report an Imaging Flow Cytometry (IFC)-based method that combines the throughput and statistical rigour of CFC with the spatial information of a microscope. By co-staining cells with Ca(2+) indicators and organelle-specific dyes we can address the spatiotemporal patterns of Ca(2+) flux in Jurkat cells after stimulation with anti-CD3. The multispectral, high-throughput nature of IFC means that the organelle co-staining functions to direct the measurement of Ca(2+) indicator fluorescence to either the endoplasmic reticulum (ER) or the mitochondrial compartments without the need to treat cells with detergents such as digitonin to eliminate cytoplasmic background. We have used this system to look at the cellular localisation of Ca(2+) after stimulating cells with CD3, thapsigargin or ionomycin in the presence or absence of extracellular Ca(2+). Our data suggest that there is a dynamic interplay between the ER and mitochondrial compartments and that mitochondria act as a sink for both intracellular and extracellular derived Ca(2+). Moreover, by generating an NFAT-GFP expressing Jurkat line, we were able to combine mitochondrial Ca(2+) measurements with nuclear translocation. In conclusion, this method enables the high throughput study of spatiotemporal patterns of Ca2(+) signals in T cells responding to different stimuli.
钙离子（Ca(2+)）是细胞信号的普遍转导因子，控制着增殖、分化、分泌和代谢等关键过程。在T细胞的背景下，通过T细胞受体的刺激已被证明可诱导细胞内储存的Ca(2+)释放。细胞质内这种突然的升高会触发质膜中离子通道的开放，允许细胞外Ca(2+)流入，进而激活关键分子如钙调磷酸酶。这一级联反应最终导致基因转录和细胞状态的改变。传统的测量Ca(2+)的方法包括分光光度法、传统流式细胞术（CFC）和活细胞成像技术。虽然每种方法都有优缺点，但没有一种能提供响应各种激动剂时Ca(2+)动员的详细情况。在这里，我们报告一种基于成像流式细胞术（IFC）的方法，该方法将CFC的通量和统计严谨性与显微镜的空间信息相结合。通过用Ca(2+)指示剂和细胞器特异性染料对细胞进行共染色，我们可以研究抗CD3刺激后Jurkat细胞中Ca(2+)通量的时空模式。IFC的多光谱、高通量特性意味着细胞器共染色的作用是将Ca(2+)指示剂荧光的测量引导至内质网（ER）或线粒体区室，而无需用洋地黄皂苷等去污剂处理细胞以消除细胞质背景。我们已经使用这个系统来观察在有或没有细胞外Ca(2+)的情况下，用CD3、毒胡萝卜素或离子霉素刺激细胞后Ca(2+)的细胞定位。我们的数据表明，内质网和线粒体区室之间存在动态相互作用，并且线粒体充当细胞内和细胞外来源的Ca(2+)的汇。此外，通过生成表达NFAT-GFP的Jurkat细胞系，我们能够将线粒体Ca(2+)测量与核转位相结合。总之，这种方法能够对T细胞中响应不同刺激的Ca2(+)信号的时空模式进行高通量研究。
