A Resorufin Derivative as a Fluorogenic Indicator for Cell Viability
A Resorufin Derivative as a Fluorogenic Indicator for Cell Viability
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试卤灵衍生物作为细胞活力的荧光指示剂
DOI：
10.2116/analsci.15.1025
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发表时间：
1999
影响因子：
1.6
通讯作者：
K. Sasamoto
中科院分区：
文献类型：
--
作者：
M. Ishiyama;Hisako Furusawa;M. Shiga;Fumio Ohseto;K. Sasamoto
作为使用放射性同位素或动物来测量活细胞数量的替代方法，越来越多地被使用。该分析快速，通常使用对细胞酶（如脱氢酶或酯酶）活性有反应的显色染料进行。3-（4,5-二甲基-2-噻唑基）-2,5-二苯基2h溴化四唑（MTT）是最常用的显色染料之一，用它可以在570 nm处比色测量脱氢酶活性（可能是线粒体的），作为细胞活力的指标然而，MTT在细胞还原时产生一种极不溶于水的甲醛染料，通常会形成沉积物，损害细胞，因此在其测定过程中需要额外的步骤来溶解甲醛。为了克服这一缺点，我们报道了一种四氮唑盐，2-（4-碘苯基）3-（4-硝基苯基）-5-（2,4-二硫苯基）- 2h四氮唑单钠盐（WST-1），它能产生高水溶性的甲醛，并被证明是一种有价值的细胞活力显色指示剂尽管WST-1已经发现了越来越多的应用，但对于大多数细胞系来说，它只能用于测量每孔（微孔板）1000到25000个细胞之间的活细胞数量。由于这些显色四氮唑盐技术对细胞活力的灵敏度有限，我们更感兴趣的是开发一种荧光法，可以检测线粒体以外的其他代谢活动作为细胞活力指标。虽然3 ',6 ' -(二- o-乙酰基-2 ',7 ' -二[N，N-二（羧甲基）氨基甲基]荧光素，四乙酰氧基甲酯（calcein - am） 5-10是一种荧光染料，可以渗透细胞膜并在细胞内酯酶水解后产生强烈的钙黄蛋白荧光，但其在515 nm处的荧光发射会受到内源性自荧光化合物的干扰。在这项工作中，我们设计了一种新的荧光化合物，7-异丁基羰基氧基- 3hphenoxazin -3-one(1，图表1)，其中间苯二酚被选为荧光团，因为它发出的荧光波长更长，并且可以在羟基上进行修饰，这对其荧光至关重要。我们还担心市售的醋酸再间萘芬在生理pH条件下相对不稳定，因此适用性有限。本文报道了1作为荧光细胞活力指示剂的合成及初步应用。
nique of increasing use as an alternative method to using radioisotopes or animals for measuring the number of viable cells. The assay is rapid and is normally performed using a chromogenic dye that responds to the activity of cellular enzymes, such as dehydrogenase or esterase. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl2H tetrazolium bromide (MTT) is among the most commonly used chromogenic dye with which dehydrogenase activity (probably of mitochondria) can be measured colorimetrically at 570 nm as an index for cell viability.1 MTT, however, produces an extremely water-insoluble formazan dye upon cellular reduction, often forming deposits which can damage the cells, and therefore an extra step is required in its assay procedure to solubilize the formazan. To overcome this disadvantage, we reported a tetrazolium salt, 2-(4-iodophenyl)3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt (WST-1), which produced highly water soluble formazan and proved to be of value as a chromogenic indicator for cell viability.2 Although WST-1 has found increasing applications3,4, it is useful only to measure the number of viable cells in the range between 1000 and 25000 cells per well (of microplates) for most cell lines. Because of the limited sensitivity for cell viability of these chromogenic tetrazolium saltbased techniques, we have been more interested in developing a fluorometric method which can detect other metabolic activities than that of mitochondria as the cell viability index. Although 3′ ,6′-(di-O-acetyl-2′ ,7′-bis[N,N-bis(carboxymethyl)aminomethyl] fluorescein, tetraacetoxymethyl ester (Calcein-AM)5–10 is such a fluorogenic dye that permeates the cell membrane and produces an intense fluorescence of calcein upon hydrolysis by intracellular esterases, its fluorescence emission at ∼ 515 nm can be interfered with endogenous auto-fluorescing compounds. In this work, we designed a new fluorogenic compound, 7-isobutyloxycarbonyloxy-3Hphenoxazin-3-one (1, Chart 1), in which resorufin was selected as the fluorophore because it fluoresces at a longer wavelength and is amenable to modification at the hydroxyl group which is essential for its fluorescence. We were also concerned that resorufin acetate, which is commercially available, was relatively unstable under physiological pH conditions, and therefore had limited applicability. This paper reports on the synthesis and the preliminary application of 1 as a fluorogenic cell viability indicator.