Kuenzel Katharina, Mofrad Sepideh Abolpour, Gilbert Daniel F
Institute of Medical Biotechnology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Paul-Gordan-Str. 3, 91052, Erlangen, Germany.
Friedrich-Alexander University (FAU) Erlangen-Nürnberg, Institute of Medical Biotechnology, Paul-Gordan-Street 3, 91052, Erlangen, Germany.
Methods Mol Biol. 2017;1601:205-214. doi: 10.1007/978-1-4939-6960-9_16.
Glycine receptor chloride channels (GlyRs) are attractive drug targets for therapeutic intervention and are also more and more recognized in the context of in vitro neurotoxicity and developmental neurotoxicity testing. Assaying the functional properties of GlyR can serve as an indicator of cellular viability and the integrity of the developing and mature central nervous system. Human pluripotent NTERA-2 (NT2) stem cells undergo neuronal differentiation upon stimulation with retinoic acid and express a large variety of neuronal proteins-including GlyR. YFP-I152L, a halide-sensitive variant of yellow fluorescent protein, allows high-throughput fluorescence-based functional analysis of GlyRs in NT2 cells. Here we describe a protocol for phenotyping of cellular viability by functional analysis of GlyR in neuronally differentiated NT2 (NT2-N) cells using YFP-I152L as a reporter of functional integrity of GlyRs. The protocol describes neuronal differentiation of NT2 stem cells, transient transfection of NT2-N cells with YFP-I152L as well as functional imaging and analysis of data from high-content imaging.
甘氨酸受体氯离子通道（GlyRs）是治疗干预中具有吸引力的药物靶点，并且在体外神经毒性和发育神经毒性测试中也越来越受到认可。检测GlyR的功能特性可作为细胞活力以及发育中和成熟的中枢神经系统完整性的指标。人多能NTERA-2（NT2）干细胞在用视黄酸刺激后会发生神经元分化，并表达多种神经元蛋白，包括GlyR。YFP-I152L是黄色荧光蛋白的卤化物敏感变体，可对NT2细胞中的GlyRs进行基于荧光的高通量功能分析。在此，我们描述了一种通过使用YFP-I152L作为GlyRs功能完整性的报告基因，对神经分化的NT2（NT2-N）细胞中的GlyR进行功能分析来对细胞活力进行表型分析的方案。该方案描述了NT2干细胞的神经元分化、用YFP-I152L对NT2-N细胞进行瞬时转染以及功能成像和来自高内涵成像的数据的分析。
