AN AUTOMATED COLORIMETRIC MICROASSAY FOR NEURONOTROPHIC FACTORS
AN AUTOMATED COLORIMETRIC MICROASSAY FOR NEURONOTROPHIC FACTORS
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DOI：
10.1016/0165-3806(86)90208-7
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发表时间：
1986-03-01
期刊：
影响因子：
--
通讯作者：
VARON, S
中科院分区：
文献类型：
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作者：
MANTHORPE, M;FAGNANI, R;VARON, S
描述了一种微量测定法，用于确定24小时后存活的神经元数量，以响应添加的神经营养因子。96孔微量滴定板中的神经元培养物提供有黄色四唑衍生物。MTT（3-（4，5-二甲基噻唑-2-基）-2，5-二苯基溴化四唑），其被活神经元选择性摄取并转化为蓝色甲瓒产物。蓝色显色的量可以使用自动微孔板分光光度计快速定量。所得的光密度与存活神经元的数量成正比。分光光度计已与计算机连接，允许打印出个体吸光度值并计算半最大值（一个营养单位）神经元存活。该测定已被用于定量的营养活动的神经生长因子和睫状神经营养因子，分别使用背根和睫状神经节神经元从8天的鸡胚。优化了测定参数，使得每天可以建立和分析约2000个神经节神经元的个体培养物，从而允许对80-120个单独的样品进行一式两份的连续滴定。神经元数量的测定和滴度计算步骤现在需要每个微板约2分钟（96个培养物），比现有方法减少了50倍的时间。
A microassay is described for determining the number of neurons surviving after 24 h in response to added neuronotrophic factors. Neuronal cultures in 96-well microtiter plates are supplied with a yellow tetrazolium derivative. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), which is taken up selectively by viable neurons and converted to a blue formazan product. The amount of blue color development can be rapidly quantified using an automatic microplate spectrotophometer. The resulting optical density is directly proportional to the number of viable neurons. The spectrophotometer has been interfaced with a computer allowing a print out of individual absorbance values and calculation of half-maximal (one trophic unit) neuronal survival. The assay has been used for the quantification of the trophic activities of nerve growth factor and ciliary neuronotrophic factor using, respectively, dorsal root and ciliary ganglionic neurons from 8-day chick embryos. Assay parameters were optimized so that about 2000 individual cultures of ganglionic neurons can be set up and anlayzed each day, thus allowing the serial titration in duplicate of 80-120 separate samples. The determination of neuronal number and titer calculation steps now requires about 2 min per microplate (96 cultures), a 50-fold reduction in time over existing methods.