In vitro micronucleus scoring by flow cytometry: Differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability
In vitro micronucleus scoring by flow cytometry: Differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability
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DOI：
10.1002/em.20170
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发表时间：
2006-01-01
影响因子：
2.8
通讯作者：
Dertinger, SD
中科院分区：
文献类型：
--
作者：
Avlasevich, SL;Bryce, SM;Dertinger, SD
体外微核试验因其在药物安全性评价和毒理学研究中的应用，近年来受到广泛关注。相对于染色体畸变分析，该测定的不那么繁琐的性质是一个驱动力，并解释了为什么许多化学和药物安全性计划已经采用了该终点。高通量微核评分系统的开发将进一步提高铅优化和其他早期药物开发工作的测定的实用性。尽管文献中描述了用于对细胞培养物衍生的微核（MN）进行评分的流式细胞术（FCM）方法的几种变化，但它们无法区分真正的MN与凋亡和坏死染色质（Nusse M和马克思K [1997]：Mutat Res 392：109-115）。在这里，我们报告这种方法的进展，即顺序染色程序被用来区别标记这些类型的子2n颗粒。使用单叠氮乙锭（EMA），标记死亡和垂死细胞的染色质。在EMA与染色质共价结合的光活化步骤后，消化细胞质膜，并将所得裂解物与RNA酶加泛核酸染料（SYTOX绿色）孵育。该过程提供了用SYTOX或SYTOX和EMA标记的游离核和亚2n颗粒的悬浮液。对热休克L5178 Y小鼠细胞的初步研究表明，EMA染色坏死和中期至晚期凋亡细胞。重要的是，连续标记程序提供了可靠的微核计数，即使当培养物中含有高百分比的死细胞。随后，进行了以下不同遗传毒物的实验：羟基脲、甲磺酸甲酯、苯并[a]芘、依托泊苷、环磷酰胺和长春碱。此外，检测了高达5 mg/ml或细胞毒性浓度的非遗传毒性物质蔗糖、三丁基锡甲醇和地塞米松。FCM数据被发现密切对应显微镜为基础的测量。总的来说，这些数据表明，这种连续EMA/SYTOX染色程序提供了可靠的，高通量的体外MN计数。
The in vitro micronucleus test has received considerable attention in recent years for its use in drug safety assessment and toxicological research. The less tedious nature of the assay relative to chromosome aberration analyses is a driving force, and explains why many chemical and drug safety programs have adopted the endpoint. Development of a high-throughput micronucleus scoring system would further enhance the utility of the assay for lead optimization and other early drug development work. Although several variations of a flow cytometric (FCM) method for scoring cell-culture-derived micronuclei (MN) have been described in the literature, they have been unable to distinguish true MN from apoptotic and necrotic chromatin (Nusse M and Marx K [1997]: Mutat Res 392: 109-115). Here, we report advances to this methodology whereby a sequential staining procedure is used to differentially label these types of sub-2n particles. With the use of ethidium monoazide (EMA), the chromatin of dead and dying cells is labeled. After a photoactivation step that covalently binds EMA to chromatin, cytoplasmic membranes are digested and resulting lysates are incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and sub-2n particles that are labeled with either SYTOX or SYTOX and EMA. Preliminary studies with heat-shocked L5178Y mouse cells demonstrated that EMA stains necrotic and mid- through late-stage apoptotic cells. Importantly, the sequential labeling procedure provided reliable micronucleus enumeration, even when cultures contained high percentages of dead cells. Subsequently, experiments with the following diverse genotoxicants were performed: hydroxyurea, methyl methanesulfonate, benzo[a]pyrene, etoposide, cyclophosphamide, and vinblastine. Additionally, the nongenotoxicants sucrose, tributyltin methoxide, and dexamethasone were tested up to 5 mg/ml, or to cytotoxic concentrations. FCM data were found to correspond closely with microscopy-based measurements. Collectively, these data suggest that this sequential EMA/SYTOX staining procedure provides reliable, high-throughput enumeration of in vitro MN.