Flow cytometry and cell sorting

Flow cytometry has become an important research tool in cytology, genetics, immunology, and microbiology. The information gained from cytometric instruments is quantitative and of high statistical precision, enabling resolution of cell subpopulations. Although increasing, application to cytology is hindered by inadequate appreciation of the nature of flow cytometry and the information obtained. Many cytologic questions can be reexamined from the perspective of this technology to obtain knowledge not accessible with conventional techniques. A flow cytometer and cell sorter are described. The physical, biochemical, and functional properties measurable by these systems are discussed.     

Recovery of RNA from flow-sorted fixed cells.

We describe a method for the preparation of RNA from ethanol-fixed cells, allowing analysis of the RNA from cells "frozen" in a given physiological state. This technique may have important applications in experiments which require prolonged cell manipulations before RNA preparation, such as investigations of cell-cycle-regulated gene expression, which require the preparation of cells for cell-cycle flow analysis, and even for long-term cell sorting. It eliminates all the inconveniences associated with the use of fresh cells, and allows cell-cycle biologists to couple flow cytometry methodology with the advancing techniques of molecular biology.     

Single cell analysis in biological oceanography and its evolutionary implications

Historically single cell analysis techniques have been usedto supplement more traditional studies of primary production.The techniques have often been used as a surrogate for microscopicanalysis and to close a gap in sea truth coverage for remotesensing and other mapping activities. In the course of developmentfrom Coulter counting to flow cytometry/cell sorting, the instrumentsand techniques have become powerful tools for allometric andataxonomic analysis as well as the quantification of pigmentsand added metabolic stains and tagged reagents. The specificquestions we ask here are: Can flow cytometry-derived data beraised to a level to discern evolutionary direction and diversity/complexity?Can we account for changes in community structure based on allometricand ataxonomic relationships across major ocean boundaries?We present evidence from different approaches and use examplesfrom flow cytometry/cell sorting that address the causes ofvariation in cell size and chlorophyll fluorescence in phytoplankton.The horizon is rapidly expanding yet questions and limitationsof ocean study persist. We believe that a road of commonalityamong oceanographers, ecologists, modelers, microbiologists,molecular biologists, physiologists and paleontologists is needed.     

Evaluation of cell numbers and viability of Saccharomyces cerevisiae by different counting methods

Four counting methods (two flow cytometry, one Coulter principle, one microscopic) are compared for measuring cell density and viability of batch-grown yeast. All gave adequate precision in measuring total cell density with no systematic difference between methods. However, the promise of flow cytometry as a rapid means of determining both quantity and quality of a cell population is shown.     

“Cytogenetic” studies of spermatids of mice carrying Cattanach's translocation by flow cytometry

The DNA content of spermatids of mice carrying Cattanach's translocation has been measured with high precision by flow cytometry. The observation that the two peaks of DNA content in the haploid region of the DNA histograms represent X-and Y-bearing spermatids was tested and confirmed. Using flow cytometry, the difference in DNA content between the X and Y chromosomes in these mice was measured to be 5.2±0.1% of the total haploid genome as compared to 3.4±0.1% in normal mice. These results demonstrated the precision of flow cytometry for cytogenetic studies. Additional information on spermatogenesis in mice bearing Cattanach's translocation was obtained and showed a gradual loss of cells during spermatogenesis in those bearing the balanced form of the translocation.     

Comparison of flow cytometry and epifluorescence microscopy for counting bacteria in aquatic ecosystems.

Flow cytometry was used to count bacterial cells from diverse origins: one strain of E. coli, one sample of lake water, and 18 samples of estuary water. To verify the accuracy and the precision of this technique, total bacteria counts made by flow cytometry were compared with counts by direct observation using epifluorescence microscopy. The results of this study showed that flow cytometry was a reliable technique for counting a mixture of bacteria in samples from aquatic ecosystems.     

Detection of Cryptosporidium parvum in soil extracts

Epifluorescent microscopy and flow cytometry were used in different combinations with fluorescein isothiocyanate-labeled immunoglobulins M and G3 to estimate the numbers of Cryptosporidium parvum oocysts in soil extracts containing 10 to 10,017 oocysts/ml. No combination had a systematic effect on accuracy or precision. Background debris may have produced overestimates at low oocyst concentrations when flow cytometry was used.     

Flow cytometry: basic principles and applications

Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell such as size and granularity simultaneously as the cell flows in suspension through a measuring device. Its working depends on the light scattering features of the cells under investigation, which may be derived from dyes or monoclonal antibodies targeting either extracellular molecules located on the surface or intracellular molecules inside the cell. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This review covers the general principles and selected applications of flow cytometry such as immunophenotyping of peripheral blood cells, analysis of apoptosis and detection of cytokines. Additionally, this report provides a basic understanding of flow cytometry technology essential for all users as well as the methods used to analyze and interpret the data. Moreover, recent progresses in flow cytometry have been discussed in order to give an opinion about the future importance of this technology.     

Peculiarities of cytometrical methods of DNA content determination in the nucleus

This work has proposed a new theoretical approach to analysis of histograms of DNA content, which are obtained by the method of flow cytometry, in cells of Drosophila melanogaster imaginal discs. The precision of measurements of the DNA amount in G₁ and G₂(M) phases has been shown to be limited by precision of instrument tuning of zero of the flow cytometer. Use of the calculative zero of the flow cytometer and of dividing cells as standards of the DNA content is able to increase severalfold the precision of the DNA measurements in nuclei of the species. Comparative analysis of errors of various methods of measurement of the DNA content in cell nuclei is also performed. For methods of flow fluorescent cytometry, confocal scanning, and cytophotometry of the Feulgen-stained nuclei, it has been shown that, at present, the mean square errors of the DNA content measurements are within the interval of values considered acceptable for biological studies (0.02 < CV < 0.06).     

Detection of Cryptosporidium parvum in Soil Extracts

Epifluorescent microscopy and flow cytometry were used in different combinations with fluorescein isothiocyanate-labeled immunoglobulins M and G3 to estimate the numbers of Cryptosporidium parvum oocysts in soil extracts containing 10 to 10,017 oocysts/ml. No combination had a systematic effect on accuracy or precision. Background debris may have produced overestimates at low oocyst concentrations when flow cytometry was used.     

Uses and future applications of flow cytometry in immunotoxicity testing.

Flow cytometry is an emerging technology that has numerous applications to immunotoxicity testing. The use and development of high-speed single-cell laser-based assays capable of quantitation of fluorescence, light scatter, and electrical impedance measurements can provide important information on xenobiotic-induced toxicity in defined target cell populations. The purpose of this article is to briefly review established and emerging immunotoxicology assays that use flow cytometry. In the coming years it is likely that many new flow cytometry assays will be developed and validated that will improve the sensitivity and perhaps specificity of immunotoxicity testing. Since flow cytometry is readily adaptable to high-throughput screening, it is also likely that this technology will increasingly find its place in the preclinical testing of drugs and chemicals in the pharmaceutical and chemical industries.     

Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

Annexin V and Sytox Green are widely used markers to evaluate apoptosis in various cell types using flow cytometry and fluorescent microscopy. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells and was validated for confocal microscopy and flow cytometry. This protocol describes simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin V and Sytox Green) by both flow cytometry and confocal microscopy in endothelial cell lines. The advantages of the described flow cytometry method over other cell-based techniques are the tremendous speed (1-2 h), exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation and apoptotic (and other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy may be very useful to reveal important spatial-temporal changes in mitochondrial superoxide production and execution of programmed cell death in virtually any cell type.     

Diversity of European spined loaches (genus Cobitis L.): an update of the geographic distribution of the Cobitis taenia hybrid complex with a description of new molecular tools for species and hybrid determination

Although the unique features of asexual reproduction and hybridization among European spined loaches (genus Cobitis ) have recently attracted the attention of conservation biologists, faunists and evolutionary biologists, the research has suffered from uncertain identification of specimens and their genomes because of the extreme morphological similarity of all the species within the hybrid complex. In this article, a Europe-wide study is reported, which was performed on samples collected by several research teams. Several complementary methodologies, such as allozyme analysis, karyotyping, flow cytometry and DNA sequencing allowed us to confirm or reject the existence of all previously reported species and their hybrids as well as to uncover several new hybrid biotypes. The biogeography of all the known biotypes, that is, parental species and hybrid biotypes, has been summarized here and the taxonomic position of two undescribed putative species mentioned in previous publications has been established. New polymerase chain reaction restriction fragment length polymorphism markers for species determination have further been developed and applied, which would allow the unambiguous identification of parental species and their genomes in the known hybrid biotypes within the complex.     

基于单克隆抗体的多元弧菌流式细胞术检测技术的研究

【目的】建立一种基于单克隆抗体的多元弧菌流式细胞仪检测技术。【方法】以副溶血弧菌表面蛋白r-OmpW的单克隆抗体(mAb)为基础,以细胞染色率为指标优化流式细胞仪检测副溶血弧菌时所需mAb的反应浓度和反应时间。通过菌落数对比评价在优化条件下流式细胞术方法的准确度、检出限和精密度。根据所建立的流式细胞术平台分析鉴定单抗对其它5种多元弧菌的特异性。【结果】流式细胞仪检测副溶血弧菌时所需r-OmpW单克隆抗体的优化反应浓度为20 mg/L,反应时间为60 min。当菌浓在104 107cells/mL范围时,检测值可信度较高,可特异性识别5种病原弧菌。对含不同菌体浓度的样品进行重复检测,变异系数均在7%以内。【结论】所建立的这种基于单克隆抗体的多元弧菌流式细胞仪检测技术可快速准确地检测多种病原弧菌。     

Plant DNA flow cytometry and estimation of nuclear genome size

BACKGROUND: DNA flow cytometry describes the use of flow cytometry for estimation of DNA quantity in cell nuclei. The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome. The nuclei are classified according to their relative fluorescence intensity or DNA content. Because the sample preparation and analysis is convenient and rapid, DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathway, and estimation of absolute DNA amount or genome size. While the former applications are relatively straightforward, estimation of absolute DNA amount requires special attention to possible errors in sample preparation and analysis. SCOPE: The article reviews current procedures for estimation of absolute DNA amounts in plants using flow cytometry, with special emphasis on preparation of nuclei suspensions, stoichiometric DNA staining and the use of DNA reference standards. In addition, methodological pitfalls encountered in estimation of intraspecific variation in genome size are discussed as well as problems linked to the use of DNA flow cytometry for fieldwork. CONCLUSIONS: Reliable estimation of absolute DNA amounts in plants using flow cytometry is not a trivial task. Although several well-proven protocols are available and some factors controlling the precision and reproducibility have been identified, several problems persist: (1) the need for fresh tissues complicates the transfer of samples from field to the laboratory and/or their storage; (2) the role of cytosolic compounds interfering with quantitative DNA staining is not well understood; and (3) the use of a set of internationally agreed DNA reference standards still remains an unrealized goal.     

Measuring the mitotic index in chemically-treated human lymphocyte cultures by flow cytometry

In the human lymphocyte chromosome aberration assay, the mitotic index (MI) is the standard cytotoxic parameter for determining which test concentrations will be evaluated for chromosome aberrations. Assessment of the MI is performed microscopically by determining the frequency of mitotic cells in a population of 1000 cells. With the commercial availability of antibodies to the mitosis-specific marker, phosphorylated-histone H3 at serine 10, automating the assessment of the MI using flow cytometry is now possible [Cytometry 32 (1998) 71]. Our laboratory has utilized and validated this technology to measure the mitotic index of chemically-treated human lymphocyte cultures. Comparisons between the microscopic and flow MI frequencies from 24h treatments with mitomycin-C, aphidicolin, eugenol, etoposide, hydroxyrurea, potassium cyanide, staurosporine, ethyl alcohol, noscapine and colcemid((R)) are presented. Our results show that the mitosis specific H3-P marker is excellent for measuring the MI frequency in human lymphocyte cultures treated up to toxic concentrations. In addition, this study demonstrates that automation of analysis by flow cytometry is an excellent alternative to the microscopic method of analysis producing less variability than the microscopic scoring and a more complete dose response curve.     

流式细胞仪的原理、应用及最新进展

流式细胞术是一种采用激光束激发单行流动的细胞,对它的散射光和携带的荧光进行探测,从而完成细胞分析和分选的技术。以流式细胞术为核心技术,流式细胞仪集光学、电子学、生物学、免疫学等多门学科和技术于一体,能够高效分析微小颗粒(如细胞,细菌)的先进科技设备。它对社会产生了深远的影响,成为了科学研究的必要工具。最近几年,流式细胞仪取得了长足进步。为了深入的了解它,本文从流式细胞仪的工作原理和技术指标,在临床医学、生物学、生殖学和制药学中的应用,以及它的世界格局、仪器功能的最新进展三方面,进行了简明、扼要的论述。展望未来:功能专业化、自动化,体积小型化,多色多参数分析能力提高和分析分选速度更快成为流式细胞仪发展的趋势。     

Cell biologists sort things out: Analysis and purification of intracellular organelles by flow cytometry

Flow cytometry was established originally for measuring DNA content and for the analysis of cell-surface markers in combination with cell sorting. During the past two decades, it has added new dimensions to various areas of immunology and medicine. Increased sensitivity and precision of flow cytometers, accompanied by the development of new fluorescent dyes and probes, has led to new applications in molecular cell biology and genetics. This article focuses on applications of flow cytometry in analysis and sorting of intracellular organelles.     

Accessing bioscience images from abstract sentences

Images (e.g., figures) are important experimental results that are typically reported in bioscience full-text articles. Biologists need to access images to validate research facts and to formulate or to test novel research hypotheses. On the other hand, biologists live in an age of information explosion. As thousands of biomedical articles are published every day, systems that help biologists efficiently access images in literature would greatly facilitate biomedical research. We hypothesize that much of image content reported in a full-text article can be summarized by the sentences in the abstract of the article. In our study, more than one hundred biologists had tested this hypothesis and more than 40 biologists had evaluated a novel user-interface BioEx that allows biologists to access images directly from abstract sentences. Our results show that 87.8% biologists were in favor of BioEx over two other baseline user-interfaces. We further developed systems that explored hierarchical clustering algorithms to automatically identify abstract sentences that summarize the images. One of the systems achieves a precision of 100% that corresponds to a recall of 4.6%.     

Use of flow cytometry in oenology to analyse yeasts

Flow cytometry is a rapid method with many microbiological applications. This technique can be used to obtain counts of viable yeasts in 30 min, whereas a 48 h incubation is necessary with plate counts. This rapid method was tested for its suitability to analyse wine yeasts in a multicentre study in our three laboratories. The study compares measurements obtained by flow cytometry and the usual method, in order to test the reliability of the new method. The results obtained were very similar in terms of both the micro-organisms detected and the precision of measurements.