WST-8方法用于蓝藻细胞活力的检测  

WST-8 Assay Method in Measuring Growth Activity of Cyanobacteria Cell

作　　者：田钰琪 周晓潮 赵妍 顾响响 赓迪 王春梅 Tian Yuqi;Zhou Xiaochao;Zhao Yan;Gu Xiangxiang;Geng Di;Wang Chunmei(School of Life Science,Beijing University of Chinese Medicine,Beijing,100029)

机构地区：[1]北京中医药大学生命科学学院

出　　处：《基因组学与应用生物学》2019年第8期3554-3558,共5页Genomics and Applied Biology

基　　金：北京中医药大学自主选题(2013-JYBZZ-JS-139)资助

摘　　要：作为重要的模式生物,蓝藻活细胞数量的测定方法应用非常广泛。本研究尝试将用于哺乳动物细胞的快速活力检测方法 WST-8方法用于蓝藻的检测。首先考察WST-8法与传统的蓝藻细胞计数法的相关性从而判断WST-8法应用的可行性,然后优化检测条件,考察检测波长、是否光照、孵育温度、孵育时间以及底物加入量对测定结果的影响。结果表明,细胞密度与WST-8方法测定的吸光度呈线性(r^2=0.996 7),此方法可用于集胞藻6803的细胞活力测定。最佳检测波长为457 nm,在25℃、30℃、37℃3个温度下,随温度升高,底物还原量增多,随孵育时间增加,产物逐渐增加,在0~120 min内呈现良好的线性关系,当底物加入量在2~10μL范围内,产物量随底物量增加而增加,当底物量大于10μL后产物量不再增加。因此,建议的检测条件为在30℃时,取100μL蓝藻培养液(浓度为1×10~7~6.4×10~7 cell/mL)于96孔板孔中,加入10μL WST-8试剂,光照恒温摇床孵育120 min,测定457 nm吸光度。As an important model organism, the assessment method in the number of cyanobacteria living cells is widely used. This study was designed to apply the WST-8 detection method in cyanobacteria which was used for quick viability measuring of the mammalian cells. Firstly, the correlation between the traditional cell counting method and the WST-8 method for cyanobacteria cells was evaluated. Secondly, incubation conditions were optimized, and the effect of detection wavelength, illumination condition, incubation time, incubation temperature and the amount of WST-8 were investigated. The results indicated that the cell density determined by the cell counting method showed a linear correlation with the absorbance determined by the WST-8 method(r^2=0.996 7).This method could be used to determine the cell viability of Synechocystis sp. PCC6803. The best detection wavelength was 457 nm. At the temperature of 25℃, 30℃, and 37℃, WST-8 substrate reduction increased with the increase of temperature. As the incubation time increased, the product gradually increased within 0~120 minutes when there was a good linear correlation. When added substrate was within the range of 2~10 μL, the amount of the product increased with the increasing amount of WST-8, and when the amount of WST-8 was more than 10 μL, the amount of the product showed no more increase. Therefore, the recommended detection steps were: transferring cyanobacteria culturing material 100 μL(cell count 1×10~7~6.4×10~7 cell/mL) to a 96-orifice plate orifice at the temperature of 30℃, then adding WST-8 reagent 10 μL, incubate it for 120 min in a shaker under conditions of light and constant temperature, and finally detecting the absorbance of 457 nm.

关 键 词：蓝藻 细胞活力 wst-8 集胞藻6803 

分 类 号：R28[医药卫生—中药学]