用流式细胞仪检测大黄鱼三倍体

通过对大黄鱼二倍体和三倍体的倍性分析,建立流式细胞仪检测三倍体的方法。大黄鱼受精卵经三倍体诱导处理后,胚胎期进行染色体滴片证实在处理组中有三倍体细胞存在。接着对该组胚胎进行育苗,获得1￣3cm的鱼苗,用流式细胞仪进行检测。以二倍体大黄鱼的肌肉组织或血液细胞DNA含量的峰值道数作为对照,用同样的方法取样处理、上机、测定处理组样本个体细胞的DNA含量的峰值道数。如果处理组个体细胞的DNA含量的峰值道数是二倍体组的1.5±0.1倍,则认为该个体为三倍体。实验结果经冷休克或静水压诱导处理的样本共检测182个,三倍体检出率为12.09%,其中有一组检出率高达55.56%。     

CD3单抗诱导小鼠胸腺细胞凋亡的流式细胞仪检测

采用不同浓度抗小鼠ＣＤ３ 复合物单抗刺激幼龄小鼠胸腺细胞,培养后分别在不同时间用流式细胞仪检测胸腺细胞的凋亡情况。结果表明,在ＣＤ３ 单抗诱导幼龄小鼠胸腺细胞４ 小时后,流式细胞仪即可测出凋亡细胞特有的ＡＰ峰。本项研究提示,用ＣＤ３ 单抗刺激未成熟胸腺细胞可以通过内源性的凋亡途径引起细胞死亡。未成熟Ｔ 细胞通过ＴＣＲ－ＣＤ３ 复合物与自身抗原接合激活上述过程可能与克隆清除的形成机制及自我耐受有关。     

流式细胞仪检测高等植物细胞核DNA含量的方法

相对于动物和微生物而言,流式细胞术在植物科学上的应用会因植物组织与细胞(如细胞壁、中央液泡、特殊细胞器等)的特殊结构以及次生代谢产物等特殊成分,造成样品在前期处理、染色及测试等方面的困难,甚至导致检测失败或结果不准确。笔者在长期运用流式细胞仪测试工作中,积累了大量的植物样本检测经验,并参考国内外相关文献,总结出从植物取材、样品制备到植物细胞核DNA流式检测的方法和技巧,可为植物科学研究者及从事流式细胞检测的技术人员提供实验参考。     

流式细胞仪在生物学中的应用

简要论述了流式细胞仪（flow cytometry,FCM）的工作原理,并对其在生物学基础科学研究中的应用进行阐述,包括对细胞凋亡、细胞周期、免疫细胞、细胞受体的研究应用。     

流式细胞仪分析T淋巴细胞亚群

用EPICS C型流式细胞仪分析微量全血法染色的T淋巴细胞亚群,关键是分辨、抓准淋巴细胞群;用Bitmap划好分析区;用阴性对照组调好LGFL的PMT和Gain后,应相对固定之,保证分析条件相一致;选好荧光二抗;尽早分析样品。     

新型多功能流式细胞仪的研制

一种新型流式细胞仪,利用一台装配有稳定的高压氙灯的落射荧光显微镜和光电倍增管以及计算机多道分析器组成的具有高分辨率和稳定性的流式细胞仪。其波长可在近紫外到近红外波段任意选择。既可用于流动样品,又可用于静止样品测定。用于流动样品测定时,喷嘴把经流体动力学方法会聚的样品流喷射到置于落射荧光显微镜物镜前方的显微镜盖玻片上,进行单细胞的快速定量分析。用于静止样品时,可用来精确测定细胞内各组分的含量。这种新型流式细胞仪可以在生物学研究和临床医学研究中得到广泛应用,有很好的发展前景。     

植物游离细胞扫描电镜样品的制备法

介绍了植物游离细胞扫描电镜样品的制备方法步骤和注意事项.     

利用流式细胞仪分选拟南芥根尖发育早期非根毛细胞

建立了应用流式细胞仪分选植物特定类型细胞的方法。以拟南芥(Arabidopsis thaliana)Wer::GFP转基因株系为材料,用激光共聚焦显微镜鉴定GFP的表达位置,采用酶解法制备拟南芥根尖原生质体,应用流式细胞仪荧光激活细胞分选技术(FACS)分选收集GFP阳性细胞,并提取细胞的RNA。结果表明,Wer::GFP转基因株系仅在根表皮发育早期的非根毛细胞中表达GFP;利用酶解法制备的根尖原生质体数目较多;从FACS分选收集的细胞中提取的RNA质量较好,可用于研究特定类型细胞的基因表达谱。应用流式细胞仪分选拟南芥非根毛细胞的方法为研究植物特定类型细胞的基因表达谱及基因功能奠定了技术基础。     

利用流式细胞仪分选拟南芥根尖发育早期非根毛细胞

建立了应用流式细胞仪分选植物特定类型细胞的方法。以拟南芥（Arabidopsis thaliana）Wer：：GFP转基因株系为材料,用激光共聚焦显微镜鉴定GFP的表达位置,采用酶解法制备拟南芥根尖原生质体,应用流式细胞仪荧光激活细胞分选技术（FACS）分选收集GFP阳性细胞,并提取细胞的RNA。结果表明,Wer：：GFP转基因株系仅在根表皮发育早期的非根毛细胞中表达GFP;利用酶解法制备的根尖原生质体数目较多;从FACS分选收集的细胞中提取的RNA质量较好,可用于研究特定类型细胞的基因表达谱。应用流式细胞仪分选拟南芥非根毛细胞的方法为研究植物特定类型细胞的基因表达谱及基因功能奠定了技术基础。     

DNA水凝胶应用于环境样品快速检测的研究进展

DNA作为生物大分子既可以引导生物发育和生命机能活动,也可以被用作构筑纳米生物材料.DNA水凝胶可以制备成兼具DNA生物功能和水凝胶特质,应用于环境样品的分析检测.依据制备DNA水凝胶长链的方法,对比分析了聚合酶链反应、杂交链式反应、滚环扩增技术的制备,物理水凝胶和化学水凝胶的合成过程和改性方法技术特点;并结合环境样品...     

Reference standards for determination of DNA content of plant nuclei

Flow cytometry was used to compare 14 potential reference standards for plant DNA content determination. Both chicken and plant internal standards were used, as were propidium iodide (PI) and 4'-6-diamidino-2-phenylindole (DAPI) as fluorochromes. Means and standard errors of the means are presented for the 14 potential reference standards, and the means are compared to those obtained by Feulgen densitometry. Five species are recommended as an initial set of international standards for future plant DNA content determinations: Sorghum bicolor cv. Pioneer 8695 (2C = 1.74 pg), Pisum sativum cv. Minerva Maple (2C = 9.56 pg), Hordeum vulgare cv. Sultan (2C = 11.12 pg), Vicia faba (2C = 26.66 pg), and Allium cepa cv. Ailsa Craig (2C = 33.55 pg). It is recommended that the reference standard of choice be one with 2C and 4C nuclear DNA content peaks similar to, but not overlapping, the 2C and 4C peaks of the target species. We recommend PI as the fluorochrome of choice for flow cytometric determination of plant DNA content. DAPI should be used only if the estimated DNA value is corroborated by using a second stain that has no bias for AT- or GC-rich sequences within genomes.     

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4',6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.     

Detection of DNA in Ancient Skeletal Remains Using DNA Flow Cytometry

Cortical bone samples were removed from individual burials from Tomb Dk31 in the Dakhleh Oasis, Egypt. The tissue was disaggregated, stained with the DNA specific fluorescent dye DAPI and analyzed using the flow cytom-eter. DNA flow cytometry measures the cellular DNA content and this is correlated with modal chromosome content. When DNA is present in skeletal remains further investigations such as extracting, amplifying and sequencing may then be carried out. The method offers a relatively rapid and inexpensive means of pinpointing samples of skeletal DNA that can be further analyzed.     

Detection of DNA in Ancient Skeletal Remains Using DNA Flow Cytometry

Cortical bone samples were removed from individual burials from Tomb Dk31 in the Dakhleh Oasis, Egypt. The tissue was disaggregated, stained with the DNA specific fluorescent dye DAPI and analyzed using the flow cytom-eter. DNA flow cytometry measures the cellular DNA content and this is correlated with modal chromosome content. When DNA is present in skeletal remains further investigations such as extracting, amplifying and sequencing may then be carried out. The method offers a relatively rapid and inexpensive means of pinpointing samples of skeletal DNA that can be further analyzed.     

流式细胞术检测胃癌前病变DNA及p21，p53含量

应用流式细胞术和免疫荧光技术,对80例胃癌前病变细胞中DNA含量,rasp21和p53蛋白进行定量检测,探讨其在胃癌前病变中,作为癌变早期标志物的临床意义.检测结果发现,胃癌前病变的DNA异倍体率随不典型增生病变的分级增高而增加, rasp21和p53蛋白的表达量亦随胃癌前病变不典型增生程度的加重而增高.DNA异倍体和rasp21表达阳性的胃癌前病变出现的癌变率显著增高,提示可能是癌变早期的分子标志物.     

Estimation of nuclear DNA content of plants by flow cytometry

A rapid and simple protocol for estimation of nuclear DNA content of plants is described. Suspensions of intact nuclei are prepared either by chopping plant tissues or lysing protoplasts in a MgSO₄ buffer, mixed with DNA standards, and stained with propidium iodide in a solution containing DNAase-free RNAase. Fluorescence intensities of the stained nuclei are measured by a flow cytometer. Values for nuclear DNA content are estimated by comparing fluorescence intensities of the nuclei of the test population with those of appropriate internal DNA standards. The same procedure can also be used for rapid determination of ploidy in plant tissues.     

Estimation of the nuclear DNA content of gossypium species

BACKGROUND AND AIMS: Gossypium is an economically important, globally distributed taxon comprising more than 50 species. DNA content estimates from about half of the species indicate over a 3-fold variation exists. However, the nine DNA content estimates for G. hirsutum reveal over a 2-fold difference for this species alone. Recent reports have shown that several plant compounds can bias DNA content estimates obtained by commonly used methods. The purpose of this research was to examine the standardization procedures used for DNA content determinations with flow cytometry as applied to Gossypium, and generate revised DNA content estimates for all available Gossypium species using best-standard practices. METHODS: Flow cytometry was used to measure fluorescence of isolated Gossypium nuclei stained with propidium iodide. Fluorescence values were converted to DNA content estimates based on the nuclear fluorescence of standard genotypes of barley, corn and rice. Various combinations of nuclei preparations relative to the standards were evaluated for their influence on the estimates. KEY RESULTS: Both external standardization and internal standardization with Oryza sativa 'IR36' yielded statistically similar DNA content estimates for Gossypium. Internal standardization with Hordeum vulgare 'Sultan' resulted in a high estimate of DNA content. Nuclear DNA content estimates were generated for 37 Gossypium species using external standardization. Estimates of ancestral genome sizes reveal that both increases and decreases in nuclear DNA content have occurred. Variation in intraspecific and intragenomic DNA content was low, and the allopolyploid AD-genome size was nearly the additive of its progenitor genomes. CONCLUSIONS: Due to unknown factors, internal standardization with H. vulgare 'Sultan' may not be appropriate for DNA content determinations of Gossypium. The current DNA content estimates support accepted cytogenetic divisions of the genus. Gossypium is a genus that exhibits genome constancy both through speciation within genomic groups and allopolyploidization.     

Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content

• Background and Aims Flow cytometry (FCM) is extensivelyused to estimate DNA ploidy and genome size in plants. In orderto determine nuclear DNA content, nuclei in suspension are stainedby a DNA-specific fluorochrome and fluorescence emission isquantified. Recent studies have shown that cytosolic compoundsmay interfere with binding of fluorochromes to DNA, leadingto flawed data. Tannic acid, a common phenolic compound, maybe responsible for some of the stoichiometric errors, especiallyin woody plants. In this study, the effect of tannic acid onestimation of nuclear DNA content was evaluated in Pisum sativumand Zea mays, which were chosen as model species. • Methods Nuclear suspensions were prepared from P. sativumleaf tissue using four different lysis buffers (Galbraith's,LB01, Otto's and Tris.MgCl₂). The suspensions were treated withtannic acid (TA) at 13 different initial concentrations rangingfrom 0·25 to 3·50 mg mL^–1. After propidiumiodide (PI) staining, samples were analysed using FCM. In additionto the measurement of nuclei fluorescence, light scatter propertieswere assessed. Subsequently, a single TA concentration was chosenfor each buffer and the effect of incubation time was assessed.Similar analyses were performed on liquid suspensions of P.sativum and Z. mays nuclei that were isolated, treated and analysedsimultaneously. FCM analyses were accompanied by microscopicobservations of nuclei suspensions. • Key Results TA affected PI fluorescence and light scatterproperties of plant nuclei, regardless of the isolation bufferused. The least pronounced effects of TA were observed in Tris.MgCl₂buffer. Samples obtained using Galbraith's and LB01 bufferswere the most affected by this compound. A newly described ‘tannicacid effect’ occurred immediately after the addition ofthe compound. With the exception of Otto's buffer, nuclei ofP. sativum and Z. mays were affected differently, with pea nucleiexhibiting a greater decrease in fluorescence intensity. • Conclusions A negative effect of a secondary metabolite,TA, on estimation of nuclear DNA content is described and recommendationsfor minimizing the effect of cytosolic compounds are presented.Alteration in light scattering properties of isolated nucleican be used as an indicator of the presence of TA, which maycause stoichiometric errors in nuclei staining using a DNA intercalator,PI.     

Flow Cytometry studies of marine phytoplankton populations

Abstract

Flow cytometry has been applied to the study of phytoplankton populations and cell components. In this work, cells cycle studies on three marine diatoms cultures were carried out by a flow cytometer. Phaeodactylum tricomutum, Chaetoceros simplex and Thalassiosira allenii, showed different DNA patterns in G1, G2, S, phases. This situation may be related to the specific algal physiology. Cultures of P. tricomutum were maintained in 4 media with different silicates concentrations. The lack of silicates did not seem produced a significative cells arresting in the cell cycle phases. During the experiment, the cell fraction in S phase, decreased in all media tested. These preliminary results could be further developed to apply flow cytometry to environmental problems in order to identify general algal groups and study algal physiology in different growth conditions.     

Simultaneous Measurement of DAPI-Sulforhodamine 101 Stained Nuclear DNA and Protein in Higher Plants by Flow Cytometry

A 2-step staining procedure is presented for simultaneous measurement of nuclear DNA and protein content in higher plants by flow cytometry. To release nuclei, plant tissues were chopped and stirred in the presence of the DNA specific fluorochrome 4', 6-diamidino-2-phenylindole (DAPI) and the nonionic detergent Triton X-100. Plant protoplasts were stirred in the DAPI dye solution with the detergent. After a short incubation period a second dye solution containing DAPI and the protein fluorochrome sulforhodamine 101 (SR 101) without detergent was added. Following another incubation, and after filtration through nylon gauze, the highly fluorescent nuclei were analyzed with an impulse cytophotometer. Accurate bivariate DNA-protein histograms were obtained with CV-values of about 2% or less for the 2C-peak of the univariate DNA parameter. The method presented here can be used for basic and applied cytogenetic studies of higher plants, for characterization of subcompartments of the cell cycle phases, or for examination of heterogeneity in plant tissues.