School of Pharmacy, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
Department of Anatomy and Medical Imaging, School of Medical Sciences, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
Sci Rep. 2021 Dec 14;11(1):23935. doi: 10.1038/s41598-021-03442-1.
Neuronal models are a crucial tool in neuroscientific research, helping to elucidate the molecular and cellular processes involved in disorders of the nervous system. Adapting these models to a high-throughput format enables simultaneous screening of multiple agents within a single assay. SH-SY5Y cells have been widely used as a neuronal model, yet commonly in an undifferentiated state that is not representative of mature neurons. Differentiation of the SH-SY5Y cells is a necessary step to obtain cells that express mature neuronal markers. Despite this understanding, the absence of a standardised protocol has limited the use of differentiated SH-SY5Y cells in high-throughput assay formats. Here, we describe techniques to differentiate and re-plate SH-SY5Y cells within a 96-well plate for high-throughput screening. SH-SY5Y cells seeded at an initial density of 2,500 cells/well in a 96-well plate provide sufficient space for neurites to extend, without impacting cell viability. Room temperature pre-incubation for 1 h improved the plating homogeneity within the well and the ability to analyse neurites. We then demonstrated the efficacy of our techniques by optimising it further for neurite outgrowth analysis. The presented methods achieve homogenously distributed differentiated SH-SY5Y cells, useful for researchers using these cells in high-throughput screening assays.
神经元模型是神经科学研究中的重要工具，有助于阐明神经系统疾病相关的分子和细胞过程。将这些模型改编为高通量格式，可以在单个测定中同时筛选多种试剂。SH-SY5Y 细胞已被广泛用作神经元模型，但通常处于未分化状态，不能代表成熟神经元。SH-SY5Y 细胞的分化是获得表达成熟神经元标志物的细胞的必要步骤。尽管有这种理解，但缺乏标准化的方案限制了分化的 SH-SY5Y 细胞在高通量测定格式中的使用。在这里，我们描述了在 96 孔板中分化和重新铺板 SH-SY5Y 细胞以进行高通量筛选的技术。在 96 孔板中以初始密度 2,500 个细胞/孔接种 SH-SY5Y 细胞，为神经突的延伸提供了足够的空间，而不会影响细胞活力。室温孵育 1 小时可提高孔内的铺板均一性和分析神经突的能力。然后，我们通过进一步优化神经突生长分析来证明我们技术的功效。所提出的方法可实现均匀分布的分化 SH-SY5Y 细胞，可用于在高通量筛选测定中使用这些细胞的研究人员。
