A guide to choosing fluorescent protein combinations for flow cytometric analysis based on spectral overlap.
A guide to choosing fluorescent protein combinations for flow cytometric analysis based on spectral overlap.
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基于光谱重叠的流式细胞仪分析选择荧光蛋白组合的指南。
DOI：
10.1002/cyto.a.23360
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发表时间：
2018-05
期刊：
影响因子：
--
通讯作者：
Grimes HL
中科院分区：
文献类型：
--
作者：
Kleeman B;Olsson A;Newkold T;Kofron M;DeLay M;Hildeman D;Grimes HL
简便的基因组工程技术的出现使得基因敲入基因表达或融合蛋白报告基因的生成变得更加容易处理。特定基因的荧光蛋白标记与表面标记分析相结合可以更具体地识别细胞群。然而，由于候选蛋白的数量和缺乏关于新荧光蛋白的更新实验数据，使用哪种荧光蛋白来生成报告构建体的问题变得困难。使这个问题更加复杂的是，大多数荧光蛋白被设计和测试用于显微镜。为了解决这个问题，我们克隆并表征了 13 种单体荧光蛋白的检测灵敏度、光谱重叠和溢出扩散，以确定在多色面板中的实用性。我们鉴定了一组五种荧光蛋白，它们具有高信噪比、最小的光谱重叠和低溢出扩散，使它们适合多色实验。具体来说，通过组合这些蛋白质中的三种来生成报告基因，即使在低水平表达下也可以进行有效的测量。由于这些蛋白质是单体，因此它们可以充当基因表达或融合蛋白报告基因。此外，随着新荧光蛋白的开发以确定其在多色面板中的有用性，这种方法可以得到推广。
The advent of facile genome engineering technologies has made the generation of knock-in gene-expression or fusion-protein reporters more tractable. Fluorescent protein labeling of specific genes combined with surface marker profiling can more specifically identify a cell population. However, the question of which fluorescent proteins to utilize to generate reporter constructs is made difficult by the number of candidate proteins and the lack of updated experimental data on newer fluorescent proteins. Compounding this problem, most fluorescent proteins are designed and tested for use in microscopy. To address this, we cloned and characterized the detection sensitivity, spectral overlap and spillover spreading of 13 monomeric fluorescent proteins to determine utility in multicolor panels. We identified a group of five fluorescent proteins with high signal to noise ratio, minimal spectral overlap and low spillover spreading making them compatible for multicolor experiments. Specifically, generating reporters with combinations of three of these proteins would allow efficient measurements even at low-level expression. Because the proteins are monomeric, they could function either as gene-expression or as fusion-protein reporters. Additionally, this approach can be generalized as new fluorescent proteins are developed to determine their usefulness in multicolor panels.
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影响因子：
3.7
作者：
Nguyen, Richard;Perfetto, Stephen;Mahnke, Yolanda D.;Chattopadhyay, Pratip;Roederer, Mario
通讯作者：
Roederer, Mario
改善绿色和红色荧光蛋白的亮度和光稳定性，用于活细胞成像和FRET报告。
影响因子：
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作者：
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通讯作者：
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作者：
Erard, Marie;Fredj, Asma;Merola, Fabienne
通讯作者：
Merola, Fabienne
影响因子：
46.9
作者：
Nagai, T;Ibata, K;Miyawaki, A
通讯作者：
Miyawaki, A