A versatile assay for the accurate, time-resolved determination of cellular viability
A versatile assay for the accurate, time-resolved determination of cellular viability
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DOI：
10.1016/s0003-2697(02)00653-x
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发表时间：
2003-03-01
影响因子：
2.9
通讯作者：
Shioi, Y
中科院分区：
文献类型：
--
作者：
Amano, T;Hirasawa, K;Shioi, Y
一个方便和通用的方法，准确，时间分辨测定细胞活力已经开发出来。常规的活力指示剂二乙酸荧光素（FDA），其在活细胞中转化为荧光化合物荧光素，被用作活力探针。使用配备有磁力搅拌器的分光荧光计测量来自细胞的荧光发射。使用该测定法，当FDA浓度小于12 μ M时，显示密度在0.5 × 10（5）至2.0 × 10（5）细胞范围内的细胞悬浮液显示线性响应。为了校准该方法，使用不同比例的活细胞和死细胞来阐述活力标准，并且烟草BY-2悬浮液的活力的相关系数计算为0.998。还发现该活力测定适用于莱茵衣藻和拟南芥培养的细胞。使用该细胞活力测定，可以进行细胞死亡的动力学分析。使用来自隐地疫霉的蛋白质激发子隐地菌素诱导烟草细胞悬浮液中的细胞死亡，死亡诱导速率的最大速度（V-max）和LD 50（半最大速度或k（1/2））的值分别计算为17.2（%死亡/h）和65 nM。(C)2003 Elsevier Science（美国）。All rights reserved.
A convenient and versatile method for the accurate, time-resolved determination of cellular viability has been developed. The conventional viability indicator fluorescein diacetate (FDA), which is converted to the fluorescent compound fluorescein in living cells, was employed as a viability probe. Fluorescence emission from cells was measured using a spectrofluorimeter equipped with a magnetic stirrer. Using this assay cell suspensions exhibiting densities in the range 0.5 x 10(5) to 2.0 x 10(5) cells displayed a linear response when FDA concentrations less than 12 muM were employed. To calibrate the method, viability standards were elaborated using different proportions of living and dead cells, and a correlation coefficient for the viability of tobacco BY-2 suspensions was calculated as 0.998. This viability assay was also found to be applicable to Chlamydomonas reinhardtii and Arabidopsis thaliana cultured cells. Using this cell viability assay, kinetic analyses of cell death could be performed. Using the proteinaceous elicitor from Phytophthora cryptogea, cryptogein, to induce cell death in tobacco cell suspensions, values for the maximum velocity of death induction rate (V-max) and the LD50 (half-maximal velocity or k(1/2)) were calculated as 17.2 (% death/h) and 65 nM, respectively. (C) 2003 Elsevier Science (USA). All rights reserved.