MacDonald Christa, Unsworth Charles P, Graham E Scott
Department of Pharmacology and Centre for Brain Research, School of Medical Sciences, The University of Auckland, Faculty of Medical and Health Sciences, Private Bag 92019, Auckland 1142, New Zealand; Department of Engineering Science, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
Department of Engineering Science, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
J Neurosci Methods. 2014 Apr 30;227:47-56. doi: 10.1016/j.jneumeth.2014.02.004. Epub 2014 Feb 14.
Human neurons (hNT neurons), obtained from the NTera2/D1 precursor cell line, are highly valued by many neuroscientists as isolation of adult human primary neuronal cells continues to elude us. hNT neurons are generated by differentiation of the NT2 precursors for a period of 4 weeks followed by 2 weeks of mitotic inhibition. This yields a heterogeneous population of neuronal phenotypes and underlying astrocyte precursors, the latter of which are very difficult to visualise using standard light microscopy. Such a mixed culture is acceptable for some applications (e.g. measurement of synaptic plasticity), whereas others (e.g. proteomics or transcriptomics) require almost pure cultures of hNT neurons.
Here we describe a simple method for obtaining highly enriched cultures of hNT neurons following the first neuronal harvest and detail several additional methods, namely flow-cytometry and xCELLigence© biosensor technology, to rapidly and reliably determine the purity and viability of the cultures.
This method of enrichment for the neurons is novel and advances the end user applications of the cells.
In addition, we apply the enrichment method to conduct analysis of cell-surface markers using flow-cytometry on the enriched neuronal cells. Furthermore, we apply this method to generate enriched neuronal cells on which we conduct analysis of cell-surface markers using flow-cytometry.
Collectively, this paper describes several new advances, which will create opportunities when using these cells and similar preparations, and provides the protocol for analysis of these cells using flow-cytometry and biosensor technology.
从NTera2/D1前体细胞系获得的人神经元（hNT神经元）受到许多神经科学家的高度重视，因为我们仍无法分离出成人原代神经元细胞。hNT神经元是通过将NT2前体分化4周，然后进行2周的有丝分裂抑制而产生的。这产生了具有不同神经元表型和潜在星形胶质细胞前体的异质群体，使用标准光学显微镜很难观察到后者。这种混合培养对于某些应用（例如突触可塑性的测量）是可以接受的，而其他应用（例如蛋白质组学或转录组学）则需要几乎纯的hNT神经元培养物。
在这里，我们描述了一种在首次收获神经元后获得高度富集的hNT神经元培养物的简单方法，并详细介绍了几种其他方法，即流式细胞术和xCELLigence©生物传感器技术，以快速可靠地确定培养物的纯度和活力。
这种神经元富集方法是新颖的，推进了细胞的最终用户应用。
此外，我们应用富集方法对富集的神经元细胞进行流式细胞术分析细胞表面标志物。此外我们应用此方法生成富集的神经元细胞，并对其进行流式细胞术分析细胞表面标志物。
总体而言，本文描述了几个新进展，这将为使用这些细胞和类似制剂创造机会，并提供了使用流式细胞术和生物传感器技术分析这些细胞的方案。
