Evaluation of hepatic subcellular fractions for Alamar blue and MTT reductase activity
Evaluation of hepatic subcellular fractions for Alamar blue and MTT reductase activity
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DOI：
10.1016/s0887-2333(01)00014-5
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发表时间：
2001-06-01
影响因子：
3.2
通讯作者：
Tarloff, JB
中科院分区：
文献类型：
--
作者：
Gonzalez, RJ;Tarloff, JB
阿利兰和MTT是用于测量培养细胞中各种化学物质的细胞毒性的指示剂。线粒体酶可还原Alcohol Blue和MTT。我们观察到增强的荧光Alcohol蓝时，肾上皮细胞与肝线粒体后上清液（S9）的馏分相比，在S9馏分的情况下培养的细胞共孵育。本研究进行，以确定是否肝细胞溶质和/或微粒体酶能够代谢阿利西蓝和/或MTT其还原产物。使用雌性Sprague-Dawley大鼠的肝脏制备S9组分以及线粒体、微粒体和细胞溶质组分。将含有1或5 mg蛋白质/ml的级分与阿利兰或MTT孵育长达4小时。测定荧光（阿利兰）或吸光度（MTT），并表示为处理的威尔斯孔和对照孔之间的差异。肝脏组分（S9、线粒体、微粒体和细胞溶质）导致Alcohol蓝荧光和MTT吸光度呈浓度和时间依赖性增加。加热可使肝S9组分对Alcohol blue和MTT的还原作用消失。肝线粒体对阿鲁纳蓝的还原作用与肝S9组分或胞质溶胶对阿鲁纳蓝的催化作用大致相当。肝线粒体对MTT的还原作用与肝S9组分或微粒体对MTT的还原作用相当。这些数据表明，线粒体，胞质和微粒体酶减少阿利兰和MTT。因此，在使用这些染料时，应谨慎地将活力降低归因于线粒体损伤。(C)2001爱思唯尔科技有限公司版权所有。
Alamar blue and MTT are indicators used to measure cytotoxicity of various chemicals in cultured cells. Both Alamar blue and MTT are reduced by mitochondrial enzymes. We observed enhanced fluorescence of Alamar blue when kidney epithelial cells were co-incubated with hepatic post-mitochondrial supernatant (S9) fractions as compared with cells incubated in the absence of S9 fractions. The present studies were carried out to determine whether hepatic cytosolic and/or microsomal enzymes were capable of metabolizing Alamar blue and/or MTT to their reduced products. Livers from female Sprague-Dawley rats were used to prepare S9 fraction, and mitochondrial, microsomal and cytosolic fractions. Fractions containing 1 or 5 mg protein/ml were incubated with Alamar blue or MTT for up to 4 h. Fluorescence (Alamar blue) or absorbance (MTT) were determined and expressed as differences between treated wells and controls. Hepatic fractions (S9, mitochondria, microsomes and cytosol) caused concentration- and time-dependent increases in Alamar blue fluorescence and MTT absorbance. Reduction of Alamar blue and MTT by hepatic S9 fraction was abolished by heating. Reduction of Alamar blue by hepatic mitochondria was approximately equivalent to that catalyzed by hepatic S9 fraction or cytosol. Reduction of MTT by hepatic mitochondria was approximately equivalent to that catalyzed by hepatic S9 fraction or microsomes. These data indicate that mitochondrial, cytosolic and microsomal enzymes reduce Alamar blue and MTT. Therefore, caution should be exercised in ascribing decreases in viability as due solely to mitochondrial damage when using either of these dyes. (C) 2001 Elsevier Science Ltd. All rights reserved.