Simplified serum- and steroid-free culture conditions for high-throughput viability analysis of primary cultures of cerebellar granule neurons.
Simplified serum- and steroid-free culture conditions for high-throughput viability analysis of primary cultures of cerebellar granule neurons.
复制标题
简化的无血清和无类固醇培养条件，用于小脑颗粒神经元原代培养物的高通量活力分析。
DOI：
10.1016/s0165-0270(01)00419-8
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发表时间：
2001
影响因子：
3
通讯作者：
Belcher,SM
中科院分区：
文献类型：
--
作者：
Wong,JK;Kennedy,PR;Belcher,SM
A serum- and steroid-free primary culture system was developed for the maintenance and automated analysis of cerebellar granule cell viability. Conventional poly-lysine coated 96-well tissue culture plates serve as a platform for growth, experimental manipulation and subsequent automated analysis of these primary cultured neurons. Cerebellar granule neurons were seeded at densities ranging from 2×104to 1.25×106cells/cm2and maintained in serum- and steroid-free culture conditions for 7 days. Viability was subsequently determined by the reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and the degree of cell death occurring over that period was determined by the release of lactate dehydrogenase (LDH). At appropriate cell densities, the results of the MTS reduction and LDH release assays were directly proportional to the initial number of cerebellar granule cells plated. Those results indicate that an initial cell density of 0.5–1.0×105cells per well (0.32 cm2) was appropriate for simultaneous analysis with the MTS reduction and LDH release assays. Both assays were then used to demonstrate the utility of this model system for analysis of tert-butyl-hydroperoxide and hydrogen peroxide induced oxidative stress. Additionally, the MTS reduction assay was used to demonstrate that the NMDA-receptor selective antagonist MK-801 was neuroprotective against glutamate-mediated excitotoxicity. This study defines a powerful and flexible primary culture system for cerebellar neurons that is useful for high-throughput analysis of factors that influence neuronal viability.
