流式细胞术在植物细胞学研究中的应用

随着细胞生物学研究从传统的定性描述发展到定量、群体的研究而形成了一门新兴的交叉学科——分析细胞学。它从定量的角度对细胞的各种形态参数与生物学特性、生化成分组成以及细胞功能等进行研究。流式细胞（FCM）分析是其主要研究手段之一。FCM是将样品细胞悬浮于液体中,在流动过程中一个一个地通过测量区进行高速测量。其特点是：快速,每秒可分析数千个细胞;统计精度高,在短时间内可获得大量的细胞信息;进行多参数相关测量;分辨率高,可检出细胞间差异的5％。目前,流式细胞术已经比较广泛地应用于动物细胞周期分析、膜脂流动性…     

流式细胞术在高等植物研究中的应用

流式细胞术(FCM)是根据所测定的各种细胞性质的不同组合,从细胞群体中把某个亚群分选出来,并对它的功能和形态学进行研究或进一步培养分析。流式细胞术具有快速、灵敏和同时进行多参数检测等优点,对其基本原理和在高等植物中的应用进行了介绍。     

流式细胞术在细胞凋亡研究中的应用

本文综述了目前使用流式细胞术研究细胞凋亡的几种方法。即Hoechst-PI染色法、选择性光解法、乙醇抽提法、吖啶橙(AO)染色法、末端标记法、缺口平移法。简要介绍了这几种方法的原理和优缺点。     

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。     

流式细胞术在哺乳动物精液质量检测中的应用

精液检测的首要目标就是快速准确地确定精子的生育力。同时具备多种特性和功能完整的精子才能受精,因而只有同时客观地检测多个指标,才能更好地反映精子的生育力。精子检测的传统方法费时费力,检测精子数量少,指标单一,而且易受操作者的主观影响,不能准确地反映精子功能。流式细胞术(FCM)为精子功能研究提供了一种快速、客观、多指标、大通量的检测手段;利用FCM检测精子的质膜完整性、顶体状态、染色质结构、线粒体功能以及细胞凋亡等,可以得知精子功能的相关情况。随着新的荧光探针、染色方法的不断开发和改进,FCM为精液质量检测提供了一种新的检测平台,应用前景极其广阔。     

荧光显微术在当代植物细胞生物学研究中的应用

自从八十年前第一次在显微镜下观察到生物组织经紫外线照射后发射荧光的现象以来,荧光显微术不断获得进步,现已发展成细胞生物学中一个重要的研究手段。高度的灵敏性和专一性、制样与观察程序的简便、尤其是适宜于活细胞研究等特点,是它所具有的独特长处。荧光显微术特别是免疫荧光技术在现代医学生物学研究中的应用是一个     

流式细胞术分析强声波对植物细胞周期的影响

应用流式细胞术分析烟草细胞在交变应力作用下细胞周期的变化。用特制的强声波发生装置产生频率和强度可调的交变应力场,研究不同频率和强度的交变应力作用后烟划细胞周期的变化,实验结果表明,在交变应力作用下直接影响细胞或细胞分裂的同步化,促进S期的DNA合成,有助于细胞有丝分裂,声波频率在400Hz至800Hz,强度在90dB到110dB内,随频率和强度的增加,交变应力使S期细胞明显增加,但频率或强度达大,反而使S期细胞大大减少。     

流式细胞术在细胞凋亡检测中的应用

凋亡是细胞受一些生理或病理信号刺激时所发生的一种程序性死亡过程.近年来,有关肿瘤细胞凋亡的研究已成为一个新的研究热点．围绕凋亡细胞出现的典型的形态变化及生化改变,建立了一些检测分析凋亡细胞的方法．文章着重概述了流式细胞术（FCM）在细胞凋亡研究中的应用,尤其是NT法、TdT法及SBIP法等新方法的价值.     

流式细胞术在水体微型生物研究中的应用

综述了流式细胞术（flow cytometry)在水体微型生物研究中的应用。包括微型生物的识别、记数和生物量研究,微型生物的细胞周期分析以及生态与生理学研究。讨论了FCM在淡水微型生物和环境生物学中的应用。FCM技术与食品的改进将促进水体微型生物的研究,从而有助于对水生生态系统的深入认识。     

流式细胞术在富营养淡水湖泊微型浮游植物细胞中的应用

应用流式细胞术(FCM)对一个富营养化淡水湖泊表、底层微型浮游植物细胞进行了初步研究。研究结果表明：流式细胞术可快速、多参数区分3种不同类群微型浮游植物。微型浮游植物细胞在表、底层占50μm以下微型颗粒物数量比例分别为21．08％、17．87％,在不同水层,微型浮游植物的优势类群及数量也不同。流式细胞术大大提高了淡水微型浮游生物研究监测水平。     

A new method for fish leucocyte counting and partial differentiation by flow cytometry

A simple and rapid method for analysis of fish blood cells is presented. Carp (Cyprinus carpio) blood was diluted 200 times with Hanks' solution containing 1 microg/ml of DiOC6(3) which is a fluorescent, lipophilic dye. After staining for 10 min, the blood cells were measured by a flow cytometer (FACS). Several blood cell populations were identified by different FL-1 (green fluorescence), FSC (forward scatter), and SSC (side scatter) properties. FL-1 v. SSC or FSC v. SSC dot-plot of stained blood cells displayed five separate cell populations: erythrocytes: a mixture of thrombocytes plus lymphocytes; monocytes; neutrophils; and basophils. The number of each type of blood cell counted by the FACS was in good agreement with those counted microscopically.     

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。     

Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species

Background and Aims: After the initial boom in the application of flow cytometryin plant sciences in the late 1980s and early 1990s, which wasaccompanied by development of many nuclear isolation buffers,only a few efforts were made to develop new buffer formulas.In this work, recent data on the performance of nuclear isolationbuffers are utilized in order to develop new buffers, generalpurpose buffer (GPB) and woody plant buffer (WPB), for plantDNA flow cytometry. Methods: GPB and WPB were used to prepare samples for flow cytometricanalysis of nuclear DNA content in a set of 37 plant speciesthat included herbaceous and woody taxa with leaf tissues differingin structure and chemical composition. The following parametersof isolated nuclei were assessed: forward and side light scatter,propidium iodide fluorescence, coefficient of variation of DNApeaks, quantity of debris background, and the number of particlesreleased from sample tissue. The nuclear genome size of 30 selectedspecies was also estimated using the buffer that performed betterfor a given species. Key Results: In unproblematic species, the use of both buffers resulted inhigh quality samples. The analysis of samples obtained withGPB usually resulted in histograms of DNA content with higheror similar resolution than those prepared with the WPB. In morerecalcitrant tissues, such as those from woody plants, WPB performedbetter and GPB failed to provide acceptable results in somecases. Improved resolution of DNA content histograms in comparisonwith previously published buffers was achieved in most of thespecies analysed. Conclusions: WPB is a reliable buffer which is also suitable for the analysisof problematic tissues/species. Although GPB failed with someplant species, it provided high-quality DNA histograms in speciesfrom which nuclear suspensions are easy to prepare. The resultsindicate that even with a broad range of species, either GPBor WPB is suitable for preparation of high-quality suspensionsof intact nuclei suitable for DNA flow cytometry.     

Quantification of plasmid loss in Escherichia coli cells by use of flow cytometry

A method was developed to study plasmid stability in Escherichia coli cells, which utilised the high speed analysis properties of flow cytometry. To discriminate between plasmid-harbouring cells and plasmid-free cells a plasmid-encoded Lac repressor protein was used to regulate the expression of a chromosomally inserted green fluorescent protein gene in the host cells. Flow cytometric analysis enabled detection and quantification of plasmid-free cells due to their green fluorescent phenotype. The reported system offers real-time analysis in combination with a very low detection level of plasmid loss in bacterial populations. This could be useful in future investigations of plasmid stability and population selection in bacterial communities.     

DNA content analysis of insect cell lines by flow cytometry

The DNA content of insect cell lines (6 lepidoptera, 1 coleoptera and 1 diptera) was determined by flow cytometry. The DNA profiles of the 8 cell lines tested were different. They were characterized by the presence of several peaks (2 to 7) corresponding to different ploidy levels, by differences in the fluorescence intensity of each peak and by the proportion of cells in each peak. Two cell lines (Cf124 and BmN) were constituted of 2 distinct populations of cells. The DNA profiles of the cell lines were stable among the passages and during the length of time culture. This technique was demonstrated to be useful for the detection of mixed cell lines and nucleopolyhedrovirus cell infection, using Autographa californica MNPV. The flow cytometry gives interesting results on the cell cycle and the ploidy level; it appears as a good tool for insect cell lines characterization. This revised version was published online in July 2006 with corrections to the Cover Date.     

A rapid method for evaluation of cell number and viability by flow cytometry

A simple, rapid and reliable method has been developed for assessing the number and viability of cells, as well as cell size, in suspension culture by the use of flow cytometry. Propidium iodide exclusion is used for viability determination and fluorescent beads serve as an internal standard for cell enumeration. The main advantages of this method are its ability to handle a large number of samples with a high degree of precision and its specificity in detecting viable cells quantitatively in a heterogeneous culture of living and dead cells and debris. The method shows only a fraction of the variation found in the haemacytometer/trypan blue counting method due to its very low operator dependence. CHO - Chinese hamster ovary; FCS - Foetal calf serum; FS - Forward scatter light; MTT - 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; NCS - newborn calf serum; PBS - Phosphate buffered saline; PI - Propidium iodide; SS - Side scatter light. This revised version was published online in July 2006 with corrections to the Cover Date.     

Use of flow cytometry with fluorescent antibodies in real-time monitoring of simultaneously inoculated alcoholic-malolactic fermentation of Chardonnay

AIMS: To monitor in real-time the changes in microbial populations and chemistry of grape juice simultaneously inoculated with Saccharomyces cerevisiae and Oenococcus oeni. METHODS AND RESULTS: Viable populations of S. cerevisiae and O. oeni in Chardonnay fermentations were identified and quantified using fluorescent dyes and fluorescently labelled antibodies in a flow cytometric assay. Fermentation chemistry was monitored using Fourier transform infrared (FTIR) spectroscopy, except for malic acid which was measured enzymatically. Malic acid utilization by O. oeni was greatest in the presence of the yeast Cepage. Growth of O. oeni was substantially slower in the presence of the yeast VL1. The three yeasts had similar fermentation rates in the presence and absence of O. oeni. CONCLUSIONS: Viable and nonviable yeast and bacterial populations can be rapidly discriminated in simultaneous malolactic-alcoholic wine fermentations using antibodies, fluorescent dyes and flow cytometry. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study using fluorescently labelled antibodies to discriminate and monitor yeast and bacterial populations in wine fermentations and offers a new approach to investigating microbial interactions in wine fermentations.     

植物原生质体在分子细胞生物学研究中的应用

植物原生质体是去除了细胞壁的裸露细胞,其具有细胞全能性,现广泛应用于植物分子细胞生物学的研究中,可以大大缩减实验周期,并有助于得到体内实验的实时检测数据。该文除了介绍植物原生质体的提取和纯化方法外,还对国内外利用各种植物的原生质体进行细胞瞬时转化、亚细胞定位、细胞融合和大分子复合物相互作用等试验进行了总结和讨论。植物原生质体还可用于基因表达模式的实时检测,并作为生物反应器的受体细胞进行代谢物的体外生产。此外,还对当前该技术所面临的瓶颈进行了分析,为植物原生质体在分子细胞生物学领域的应用提供帮助,为技术的优化和推广提供参考。     

Analysis of DNA distributions from flow cytometry

A recently proposed automatic procedure for analyzing DNA distribution from flow cytometric data is extensively tested against simulated data. After a discussion of the procedure itself and of the simulation program, the results obtained are reported. They are evidence of the reliability of the procedure in extracting the proper underlying DNA distribution from sets of data obtained under various simulated instances. The different sources of error are then analyzed, along with their quantitative effects on the fit of the fluorescence histogram.