人类外周血淋巴细胞微核测定方法的改进

微核试验是70年代由Schmid建立起来 的一种检测体内染色体畸变的方法。此法简 便、迅速、也较敏感。现广泛用于筛选化学致突 变剂和辐射损伤的研究。微核试验一般多以小 鼠骨髓为试验材料,但也可以检测人类外周血 淋巴细胞的微核。     

重铬酸钾对蚕豆根尖细胞致畸效应的研究

以蚕豆根尖为材料,研究重铬酸钾对蚕豆根尖细胞的致畸效应。采用蚕豆根尖细胞的微核试验和染色体畸变试验方法,以不同浓度的重铬酸钾为诱变剂,测定蚕豆根尖细胞的微核率和染色体畸变率。结果表明:重铬酸钾能诱发较高频率的微核率,即在一定浓度范围内,其微核率随重铬酸钾处理浓度的升高而增加,但高于一定浓度后反而呈下降趋势;不同浓度的重铬酸钾均使蚕豆根尖细胞有丝分裂指数增大;重铬酸钾还能诱导蚕豆根尖细胞产生较高频率的染色体畸变,且产生多种类型的染色体畸变。结论是重铬酸钾对蚕豆根尖细胞具有明显的致畸效应。     

褐藻寡糖抗环磷酰胺诱导蚕豆根尖的细胞遗传毒性

采用蚕豆根尖细胞的微核试验和染色体畸变试验方法,测定不同浓度的褐藻寡糖对环磷酰胺(cyclophosphamide,CP)诱导的蚕豆根尖细胞的微核率、有丝分裂指数和染色体畸变率的影响。结果表明:褐藻寡糖能有效抑制环磷酰胺诱导的蚕豆根尖细胞微核的产生,即在一定浓度范围内,微核率随褐藻寡糖处理浓度的降低而减少,但低于一定浓度后反而呈上升趋势;不同浓度的褐藻寡糖均可使蚕豆根尖细胞有丝分裂指数增大;褐藻寡糖还能有效降低蚕豆根尖细胞染色体畸变率。因此,褐藻寡糖对蚕豆根尖细胞具有明显的诱抗活性和调节细胞分裂生长的效应。     

用微核试验测定几种茶树昆虫病毒对小白鼠的安全性

用一种快速简便的测定细胞染色体是否被损伤的方法(即微核试验)测定茶毛虫NPV、油茶尺蠖NPV及油桐尺蠖NPV对小白鼠的安全性,通过测定其骨髓中嗜多染红细胞微核的出现频率,证明无论是单独或是混合感染方式均不能测出这些病毒对骨髓细胞染色体有特定的损伤,而阳性对照环磷酰胺所诱发的微核率与其相比极为显著。此法可作为评价昆虫病毒对哺乳动物安全性的一个重要方法。     

乙酸铜对蚕豆根尖细胞致畸效应

采用蚕豆根尖细胞的微核试验和染色体畸变试验方法,以不同浓度的乙酸铜为诱变剂,选择不同的处理时间,测定蚕豆根尖细胞的有丝分裂指数、微核率和染色体畸变率。结果表明：乙酸铜能诱发较高频率的微核率,处理6h、12h时微核率均随着乙酸铜浓度的升高而增加,具有明显的剂量效应;处理24h时在实验浓度范围内,其微核率随乙酸铜浓度的升高而增加,但高于一定浓度后反而呈下降趋势。不同浓度的乙酸铜在不同处理时间均使蚕豆根尖细胞有丝分裂指数增大。乙酸铜还能诱导蚕豆根尖细胞产生较高频率的染色体畸变,且产生多种类型的染色体畸变。因此,乙酸铜对蚕豆根尖细胞具有明显的致畸效应。     

丫啶橙荧光染色法在微核试验中的应用

染色体或染色单体断裂后所遗留下来的断 片可通过细胞分裂在一定时期内保留于子细胞 中;纺锤体细胞器功能失调也可形成断片;这些 断片称为微核。小鼠体内的微核试验就是利用 微核来观察染色体畸变的另一种表现形式。由 于Giemsa染色法简单,快速、经济、因而被广 泛采用。但Giemsa染色法特异性较差,非特 异着色颗粒有时很难与微核相区别,影响结果 的判断,为此有必要建立一种更加特异的观察 方法。近年来荧光显微技术在细胞学、免疫学、 微生物学等多个领域中得到广泛应用,我们参 照有关文献〔1-4,将py咤橙(Acridine Orange)荧 光染色法应用于微核试验,并获满意的结果。     

文蛤肉水解液的致突变性

目的评价文蛤肉水解液的致突变性,为文蛤的开发利用提供实验数据。方法采用Ames试验、小鼠嗜多染红细胞骨髓微核试验和CHL细胞体外染色体畸变试验3项致突变性试验,检测文蛤肉水解液有无致突变作用。结果 Ames试验中文蛤肉水解液各剂量组回变菌落数均在正常范围内,均未超过自发回变菌落数的2倍,在加与不加S9时5株试验菌株结果为阴性。CHL细胞体外染色体畸变试验,文蛤肉水解液各剂量组的染色体畸变率与阴性对照组比较,差异无显著性(P0.05),结果为阴性。小鼠骨髓微核试验,各剂量组的微核率与阴性对照组比较,差异无显著性(P0.05),结果为阴性。结论在本试验条件和范围下,文蛤肉水解液未见潜在的致突变作用。     

硫酸铜对蚕豆根尖细胞有丝分裂的影响

以蚕豆根尖为材料,研究硫酸铜对蚕豆根尖细胞的遗传毒性效应。采用蚕豆根尖细胞的微核试验方法和染色体畸变试验方法,以不同浓度的硫酸铜为诱变剂,测定蚕豆根尖细胞的有丝分裂指数、微核率和染色体畸变率。结果表明:不同浓度的硫酸铜均能使蚕豆根尖细胞有丝分裂指数明显增加,即5个实验组的分裂指数均明显高于对照组(P<0.01或P<0.001);不同浓度的硫酸铜对蚕豆根尖细胞有丝分裂各期百分数的影响有异;能诱发较高频率的微核率,即在一定浓度范围内,其微核率随硫酸铜处理浓度的升高而增加,但随着硫酸铜浓度的进一步升高而呈下降趋势;硫酸铜还能诱导染色体产生多种类型的畸变,染色体畸变率随硫酸铜处理浓度的升高而增加,随着硫酸铜浓度的进一步升高而呈下降趋势,但均明显高于对照组(P<0.001)。结论是硫酸铜对蚕豆根尖细胞具有明显的遗传毒性效应。     

氟化钠致突变作用的研究

以昆明种大鼠和小鼠分别进行氟化钠诱发染色体畸变和微核试验,结果表明,在本实验条件下,未 见组化钠诱发动物骨髓细胞染色体畸变率及微核率明显增高。     

浊漳河水体污染物对蚕豆根尖细胞的遗传毒性研究

运用蚕豆根尖细胞微核试验技术对浊漳河南段6个代表性样点水体中有机污染物的遗传毒性进行了研究,为浊漳河流域水体污染状况的有效监测提供理论依据.结果显示:(1)浊漳河南段各监测断面的水体有机污染物对蚕豆根尖细胞均产生了不同程度的损伤,表现出细胞微核、断片、染色体桥等多种异常形态,严重时导致细胞坏死和细胞凋亡.(2)6个样点水样处理的蚕豆根尖细胞微核率在21.47‰～64.77‰,极显著高于对照组(P<0.01).(3)各样点水样对蚕豆根尖细胞的遗传损伤程度依次为:王桥>店上>漳泽水库>北寨>黄碾>五阳;在有丝分裂后期,蚕豆根尖中异常细胞最高达70%,且异常细胞比率有随着污染程度增大而升高的趋势.研究表明,蚕豆根尖细胞微核是水质毒理检测的有效指标,其细胞有丝分裂后期异常细胞比率也可作为水质毒理检测的观察指标.     

Flow cytometric analysis of micronucleated reticulocytes in mouse bone marrow

This laboratory has previously reported a flow cytometric procedure for quantitatively analyzing mouse peripheral blood reticulocytes for micronucleus content. The current study extends this line of investigation by evaluating whether these same flow cytometric scoring procedures can be applied to the analysis of mouse bone marrow samples. To validate the method, three groups of male BALB/c mice were treated with 100 mg/kg b.wt. methyl methanesulfonate. Bone marrow samples were collected 20, 40 or 60 h after administration. A set of 5 untreated animals was included to provide an indication of spontaneous micronucleus frequencies. The cells were fixed with ultracold methanol, treated with ribonuclease, and labeled with anti-CD71 antibody (FITC conjugate) and propidium iodide. This fixing and labeling procedure resulted in the resolution of the micronucleated reticulocyte population and facilitated high-speed acquisition and enumeration via flow cytometry. The number of micronucleated reticulocytes was determined flow cytometrically by the analysis of 10?000 total reticulocytes per bone marrow sample. In addition to these automated measurements, slides stained with acridine orange were prepared and the number of micronuclei per 1000 reticulocytes was determined microscopically for each sample. The resulting data demonstrate that flow cytometry can effectively enumerate micronucleated reticulocytes in mouse bone marrow. The advantages associated with an objective, high throughput scoring methodology are also clearly indicated.     

Comparison of methods for assessing integrity of equine sperm membranes

Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Bland-Altman methodology. Eosin-nigrosin staining yielded higher (2%) overall mean values for SMI than did flow cytometry. Flow cytometry yielded higher (6%) overall mean values for SMI than did the automated cell counter. As percentage of membrane-damaged sperm increased, agreement of SMI measurement between methods decreased. When semen contained 50-79% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = −26.9 to 24.3%; i.e., a 51.2% span) than for SMI determined by flow cytometry and the automated cell counter (range = −3.1 to 17.0%; 20.1% span). When sperm populations contained <50% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = −35.9 to 19.0%; 54.9% span) than for SMI determined by flow cytometry and the automated cell counter (range = −11.6 to 28.7%; 40.3% span). We concluded that eosin-nigrosin staining assessments of percent membrane-intact sperm agreed less with flow cytometry when <80% of sperm had intact membranes, whereas automated cell counter assessments of percent membrane-intact sperm agreed less with flow cytometry when <30% of sperm had intact membranes.     

Methods of automated cell analysis and their application in radiation biology

The present review is concerned with the methods of automated analysis of biological micro-objects and covers two groups into which all the systems of automated analysis can be divided--systems of flow (flow cytometry) and scanning (image analysis systems) type. Particular emphasis has been placed on their use in radiobiological studies, namely, in the micronucleus test, a cytogenetic assay for monitoring the clastogenic action of ionizing radiation commonly used at present. It is evident that the problem is acute, with of radiobiologists' interest in the biological action of low-dose radiation recently increasing. In addition, the estimation of a low-level damage requires the analysis of a large number of experimental objects. Examples of using both the methods elsewhere and actual setups are given. The analysis of advantages and disadvantages of the methods of automated cell analysis enables us to choose more thoroughly between the systems of flow and scanning type to use them in particular research.     

Automated analysis of the cytokinesis-block micronucleus assay for radiation biodosimetry using imaging flow cytometry

The cytokinesis-block micronucleus (CBMN) assay is employed in biological dosimetry to determine the dose of radiation to an exposed individual from the frequency of micronuclei (MN) in binucleated lymphocyte cells. The method has been partially automated for the use in mass casualty events, but it would be advantageous to further automate the method for increased throughput. Recently, automated image analysis has been successfully applied to the traditional, slide-scoring-based method of the CBMN assay. However, with the development of new technologies such as the imaging flow cytometer, it is now possible to adapt this microscope-based assay to an automated imaging flow cytometry method. The ImageStream^X is an imaging flow cytometer that has adequate sensitivity to quantify radiation doses larger than 1 Gy while adding the increased throughput of traditional flow cytometry. The protocol and analysis presented in this work adapts the CBMN assay for the use on the ImageStream^X. Ex vivo-irradiated whole blood samples cultured for CBMN were analyzed on the ImageStream^X, and preliminary results indicate that binucleated cells and MN can be identified, imaged and enumerated automatically by imaging flow cytometry. Details of the method development, gating strategy and the dose response curve generated are presented and indicate that adaptation of the CBMN assay for the use with imaging flow cytometry has potential for high-throughput analysis following a mass casualty radiological event.     

Cellometer image cytometry as a complementary tool to flow cytometry for verifying gated cell populations

Traditionally, many cell-based assays that analyze cell populations and functionalities have been performed using flow cytometry. However, flow cytometers remain relatively expensive and require highly trained operators for routine maintenance and data analysis. Recently, an image cytometry system has been developed by Nexcelom Bioscience (Lawrence, MA, USA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Image cytometry is analogous to flow cytometry in that gating operations can be performed on the cell population based on size and fluorescent intensity. In addition, the image cytometer is capable of capturing bright-field and fluorescent images, allowing for the measurement of cellular size and fluorescence intensity data. In this study, we labeled a population of cells with an enzymatic vitality stain (calcein-AM) and a cell viability dye (propidium iodide) and compared the data generated by flow and image cytometry. We report that measuring vitality and viability using the image cytometer is as effective as flow cytometric assays and allows for visual confirmation of the sample to exclude cellular debris. Image cytometry offers a direct method for performing fluorescent cell-based assays but also may be used as a complementary tool to flow cytometers for aiding the analysis of more complex samples.     

Fundamentals of flow cytometry

Flow cytomelers arc instruments that arc used primarily to measure the physical and biochemical characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents, such as nuclei and organelles. Flow cytomcters are investigative tools for a broad range of scientific disciplines because they make measurements on thousands of individual cells/ particles in a matter of seconds. This is a unique advantage relative to other detection instruments that provide bulk particle measurements. Flow cytomety is a complex and highly technical field; therefore, a basic understanding of the technology is essential for all users. The purpose of this article is to provide fundamental information about the instrumentation used for flow cytometry as well as the methods used to analyze and interpret data. This information will provide a foundation to use flow cytometry effectively as a research tool.     

Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay

An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Mol. Mutagen. 47 (2006) 56–66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei—the genotoxicants mitomycin C (MMC), etoposide (ETOPO), and vinblastine (VB), and the non-genotoxicants sucrose (SUC), staurosporine (STS), and dexamethasone (DEX). The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow™ kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24 h over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy- and flow cytometry-based scoring methods detected concentration-dependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple flow cytometric technique, and that the scoring system is transferable across laboratories. Furthermore, a concurrent assessment of cytotoxicity, Flow-NBR, may help reduce the occurrence of irrelevant positive results, as it may represent a more appropriate means for choosing top concentration levels. Finally, the data presented herein reinforce concerns about the manner in which cytotoxicity limits are described in guidance documents, since these recommendations tend to cite fixed cut-off values without reference to methodology.     

In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity

This laboratory has previously reported on the development of a flow cytometry-based method for scoring in vitro micronuclei in mouse lymphoma (L5178Y) cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Molec. Mutagen. 47 (2006) 56–66]. With this method, necrotic and mid/late stage apoptotic cells are labeled with the fluorescent dye ethidium monoazide. Cells are then washed, stripped of their cytoplasmic membranes, and incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and micronuclei that are differentially stained relative to chromatin associated with dead/dying cells. The current report extends this line of investigation to include the human cell line TK6. Additionally, methods are described that facilitate simultaneous quantitative analysis of cytotoxicity, perturbations to the cell cycle, and what we hypothesize is aneuploidization. This comprehensive cytogenetic damage assay was evaluated with the following diverse agents: etoposide, ionizing radiation, methyl methanesulfonate, vinblastine, ethanol, and staurosporine. Cells were harvested after 30 h of continuous treatment (in the case of chemicals), or following graded doses of radiation up to 1 Gy. Key findings include the following: (1) Significant discrepancies in top dose selection were found for five of the six agents studied when relative survival measurements were based on Coulter counting versus flow cytometry. (2) Both microscopy- and flow cytometry-based scoring methods detected dose-dependent micronucleus formation for the four genotoxic agents studied, whereas no significant increases were observed for the presumed non-genotoxicants ethanol and staurosporine when top dose selection was based on flow cytometric indices of cytotoxicity. (3) SYTOX and ethidium monoazide fluorescence signals conveyed cell cycle and cell death information, respectively, and appear to represent valuable aids for interpreting micronucleus data. (4) The frequency of hypodiploid nuclei increased in response to each of the genotoxic agents studied, but not following exposure to ethanol or staurosporine. Collectively, these results indicate that a comprehensive assessment of genotoxicity and other test article-induced toxicities can be acquired simultaneously using a simple two-color flow cytometry-based technique.     

A novel image-based cytometry method for autophagy detection in living cells

Autophagy is an important cellular catabolic process that plays a variety of important roles, including maintenance of the amino acid pool during starvation, recycling of damaged proteins and organelles, and clearance of intracellular microbes. Currently employed autophagy detection methods include fluorescence microscopy, biochemical measurement, SDS-PAGE and western blotting, but they are time consuming, labor intensive, and require much experience for accurate interpretation. More recently, development of novel fluorescent probes have allowed the investigation of autophagy via standard flow cytometry. However, flow cytometers remain relatively expensive and require a considerable amount of maintenance. Previously, image-based cytometry has been shown to perform automated fluorescence-based cellular analysis comparable to flow cytometry. In this study, we developed a novel method using the Cellometer image-based cytometer in combination with Cyto-ID^® Green dye for autophagy detection in live cells. The method is compared with flow cytometry by measuring macroautophagy in nutrient-starved Jurkat cells. Results demonstrate similar trends of autophagic response, but different magnitude of fluorescence signal increases, which may arise from different analysis approaches characteristic of the two instrument platforms. The possibility of using this method for drug discovery applications is also demonstrated through the measurement of dose-response kinetics upon induction of autophagy with rapamycin and tamoxifen. The described image-based cytometry/fluorescent dye method should serve as a useful addition to the current arsenal of techniques available in support of autophagy-based drug discovery relating to various pathological disorders.     

A novel image-based cytometry method for autophagy detection in living cells

Autophagy is an important cellular catabolic process that plays a variety of important roles, including maintenance of the amino acid pool during starvation, recycling of damaged proteins and organelles, and clearance of intracellular microbes. Currently employed autophagy detection methods include fluorescence microscopy, biochemical measurement, SDS-PAGE and western blotting, but they are time consuming, labor intensive, and require much experience for accurate interpretation. More recently, development of novel fluorescent probes have allowed the investigation of autophagy via standard flow cytometry. However, flow cytometers remain relatively expensive and require a considerable amount of maintenance. Previously, image-based cytometry has been shown to perform automated fluorescence-based cellular analysis comparable to flow cytometry. In this study, we developed a novel method using the Cellometer image-based cytometer in combination with Cyto-ID^® Green dye for autophagy detection in live cells. The method is compared with flow cytometry by measuring macroautophagy in nutrient-starved Jurkat cells. Results demonstrate similar trends of autophagic response, but different magnitude of fluorescence signal increases, which may arise from different analysis approaches characteristic of the two instrument platforms. The possibility of using this method for drug discovery applications is also demonstrated through the measurement of dose-response kinetics upon induction of autophagy with rapamycin and tamoxifen. The described image-based cytometry/fluorescent dye method should serve as a useful addition to the current arsenal of techniques available in support of autophagy-based drug discovery relating to various pathological disorders.