Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Aachen, Germany.
Institute of Molecular Biotechnology (Biology VII), RWTH Aachen University, Aachen, Germany.
PLoS One. 2020 Dec 3;15(12):e0243286. doi: 10.1371/journal.pone.0243286. eCollection 2020.
In vitro and ex vivo development of novel therapeutic agents requires reliable and accurate analyses of the cell conditions they were preclinical tested for, such as apoptosis. The detection of apoptotic cells by annexin V (AV) coupled to fluorophores has often shown limitations in the choice of the dye due to interference with other fluorescent-labeled cell markers. The SNAP-tag technology is an easy, rapid and versatile method for functionalization of proteins and was therefore used for labeling AV with various fluorophores. We generated the fusion protein AV-SNAP and analyzed its capacity for the specific display of apoptotic cells in various assays with therapeutic agents. AV-SNAP showed an efficient coupling reaction with five different fluorescent dyes. Two selected fluorophores were tested with suspension, adherent and peripheral blood cells, treated by heat-shock or apoptosis-inducing therapeutic agents. Flow cytometry analysis of apoptotic cells revealed a strong visualization using AV-SNAP coupled to these two fluorophores exemplary, which was comparable to a commercial AV-Assay-kit. The combination of the apoptosis-specific binding protein AV with the SNAP-tag provides a novel solid method to facilitate protein labeling using several, easy to change, fluorescent dyes at once. It avoids high costs and allows an ordinary exchange of dyes and easier use of other fluorescent-labeled cell markers, which is of high interest for the preclinical testing of therapeutic agents in e.g. cancer research.
在体外和体内开发新型治疗药物时，需要对它们进行临床前测试的细胞条件进行可靠和准确的分析，如细胞凋亡。通过与荧光染料偶联的膜联蛋白 V（AV）检测凋亡细胞，由于与其他荧光标记的细胞标记物的干扰，常因染料选择而受到限制。SNAP 标签技术是一种用于蛋白质功能化的简便、快速且通用的方法，因此被用于用各种荧光染料标记 AV。我们生成了 AV-SNAP 融合蛋白，并分析了其在各种治疗药物的检测中对凋亡细胞的特异性显示能力。AV-SNAP 与五种不同的荧光染料发生了有效的偶联反应。选择两种荧光染料用于检测悬浮、贴壁和外周血细胞，这些细胞经热休克或凋亡诱导治疗药物处理。用流式细胞术分析凋亡细胞，结果显示，用这两种荧光染料偶联的 AV-SNAP 可有效地对其进行可视化，这与商业 AV-Assay 试剂盒相当。凋亡特异性结合蛋白 AV 与 SNAP 标签的结合为使用多种、易于改变的荧光染料同时进行蛋白质标记提供了一种新的固体方法。它避免了高昂的成本，并且允许染料的普通交换和其他荧光标记的细胞标记物的更简单使用，这对于例如癌症研究中的治疗药物的临床前测试具有重要意义。
