Interference in MTT cell viability assay in activated macrophage cell line
Interference in MTT cell viability assay in activated macrophage cell line
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DOI：
10.1016/s0003-2697(02)00631-0
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发表时间：
2003-02-15
影响因子：
2.9
通讯作者：
Giovine, M
中科院分区：
文献类型：
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作者：
Pozzolini, M;Scarfi, S;Giovine, M
MTT 1（3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四唑溴化物）试验是一种简单的比色法，用于测量细胞毒性、增殖或细胞活力，最早由Mosmann于1983年开发[1]。MTT是一种黄色的水溶性四氮唑盐。代谢活性细胞能够通过四唑环的还原裂解将这种染料转化为不溶于水的深蓝色甲瓒[1]。然后，可以通过测量溶液在570 nm处的吸光度来溶解和定量甲瓒晶体，并且所得值与活细胞的数量相关。通过使用96孔微量滴定板和多孔分光光度计，该测定可以半自动化地处理大量样品并提供细胞数量的快速测量。因此，该方法在过去十年中主要用于人和哺乳动物细胞系的抗癌药物筛选试验[2-4]，并通过几种细胞系统中的比较试验进一步验证[3]。MTT还原中涉及的细胞反应尚未完全了解，但线粒体琥珀酸脱氢酶系统似乎主要涉及[1，5]。然而，目前还没有令人信服的证据表明线粒体是完整细胞中MTT还原的唯一位点，此外，一些作者观察到，当该测定法用于干扰素处理的肺癌细胞系时，可能出现伪影[6]。本工作的目的是研究MTT测定法在活化的巨噬细胞系上的可靠性，验证活化的巨噬细胞代谢对MTT还原的可能干扰。使用鼠巨噬细胞系RAW 264.7和卵巢癌细胞系HeLa作为对照细胞进行实验。将MTT细胞活力试验与SYTOX绿色核酸染色法进行比较。该方法基于阳离子非对称膜不渗透花青染料，其通常用于对具有膜损伤的死细胞进行染色[7]。染料在与细胞核酸结合后产生荧光信号[8]。在这里，我们采用这种方法来定量96孔微量滴定板上的活贴壁细胞，之前去除死细胞和适当的透化后。选择SYTOX绿色方法作为对照试验，因为其基于完全不同的定量方法。事实上，虽然MTT过程取决于酶细胞活性，其可能在不同的细胞阶段发生变化，但核酸染色与细胞核含量简单地成比例相关，从而避免了代谢产物的干扰。
The MTT1 (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay is a simple colorimetric method to measure cytotoxicity, proliferation, or cell viability first developed by Mosmann in 1983 [1]. MTT is a yellow, water-soluble, tetrazolium salt. Metabolically active cells are able to convert this dye into a water-insoluble dark blue formazan by reductive cleavage of the tetrazolium ring [1]. Formazan crystals, then, can be dissolved and quantified by measuring the absorbance of the solution at 570 nm, and the resultant value is related to the number of living cells. By using 96-well microtiter plates and a multiwell spectrophotometer this assay can be semiautomated to process a large number of samples and provide a rapid measurement of cell number. Therefore this method has been mainly used in the past decade for anticancer drug screening assays on human and mammalian cell lines [2–4], further validated by comparative assays in several cellular systems [3]. The cellular reactions involved in MTT reduction are not completely understood, but the mitochondrial succinate dehydrogenase system seems to be primarily involved [1, 5]. Nevertheless, there is currently no convincing evidence that the mitochondria are the only site of MTT reduction in the intact cell and in addition some authors have observed the possible appearance of artifacts when the assay is used on a lung cancer cell line treated with interferon [6].The aim of this work was to study the reliability of the MTT assay on activated macrophage cell line, to verify the possible interference of metabolism of activated macrophages on MTT reduction. Experiments were performed using the murine macrophage cell line RAW 264.7 and the ovarian carcinoma cell line HeLa as control cells. MTT cell viability tests were compared to the SYTOX Green nucleic acid stain method. This method is based on a cationic nonsymmetrical membrane-impermeant cyanine dye that is normally used to stain dead cells with membrane damage [7]. The dye develops a fluorescent signal after binding to cellular nucleic acids [8]. Here we employed this method to quantitate living adherent cells on 96-well microtiter plates, previous removal of dead cells and after the opportune permeabilization. The SYTOX Green method was chosen as the control assay because it is based on a completely different quantification approach. In fact, while the MTT process depends on enzymatic cellular activities that could be subjected to alterations during different cellular phases, the nucleic acid stain is simply proportionally related to the cell nuclear content, thus avoiding the interference by metabolic artifacts.