Ng Neville S, Ooi Lezanne
Illawarra Health and Medical Research Institute, Wollongong, New South Wales, Australia.
Bio Protoc. 2021 Jan 5;11(1):e3877. doi: 10.21769/BioProtoc.3877.
2',7'-dichlorofluorescein (DCF) and derivatives are commonly used as fluorescent indicators of a broad spectrum of reactive oxygen species (ROS) generation in cell-based assays. However, there are numerous challenges inherent to the utilization of DCF probes for intracellular microscopic analysis, including photostability and probe efflux. Plate spectroscopy is comparatively simple and scalable compared to microscopy or flow cytometry-based acquisition, however is often subject to artefacts, including those introduced by thermal gradients and normalization methods. In this protocol we demonstrate a simple and sensitive plate spectrometry-based protocol utilizing the probes HDCFDA and sulforhodamine B. The rapid sulforhodamine B assay (SRB) for cellular protein allows for a stable endpoint measurement of total cell population while also preserving morphology, can be combined or run in parallel with any other assay for normalization of readout to cell mass, and complemented by microscopic scoring of cell number and nuclear count. The oxidative stress and normalisation methods may enhance fields of research investigating cell differentiation, stress, or toxicity.. .
2',7'-二氯荧光素（DCF）及其衍生物通常用作基于细胞的分析中广泛活性氧（ROS）生成的荧光指示剂。然而，将DCF探针用于细胞内显微镜分析存在许多固有挑战，包括光稳定性和探针外排。与基于显微镜或流式细胞术的采集相比，平板光谱相对简单且可扩展，然而它经常受到假象的影响，包括由热梯度和归一化方法引入的假象。在本方案中，我们展示了一种基于平板光谱法的简单且灵敏的方案，该方案使用探针HDCFDA和磺酰罗丹明B。用于细胞蛋白质的快速磺酰罗丹明B测定（SRB）能够对总细胞群体进行稳定的终点测量，同时还能保留细胞形态，可以与任何其他用于将读数归一化至细胞质量的测定相结合或并行运行，并通过细胞数量和核计数的显微镜评分进行补充。氧化应激和归一化方法可能会加强对细胞分化、应激或毒性的研究领域。
