Early Features of Apoptosis Detected by Four Different Flow Cytometry Assays

The objective of this study was to investigate the sensitivity, specificity and reproducibility of some frequently used apoptosis assays. The degree of apoptosis was tested in two T-lymphoblastoid cell lines, HSB and Jurkat, in which apoptosis was induced by ionizing radiation. HSB and Jurkat samples were taken before, and 0, 2, 4, 6, 8 and 24 h after irradiation with 6 and 10 Gray, or with 10 and 14 Gray, respectively. Four frequently used flow cytometric techniques were evaluated: (i) Annexin V/Propidium Iodide assay, detecting the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, simultaneously with preservation of the membrane integrity; (ii) Terminal deoxynucleotidyl Transferase (TdT) Uridine triphosphate (UTP) nick end labelling (TUNEL), revealing the presence of DNA strand breaks; (iii) DNA-flow cytometry, measuring DNA-stainability (DNA-fragmentation assay) and (iv) Phycoerythrin-labelled (PE) Apo2.7-assay, a monoclonal antibody against 7A6 antigen, a protein, which becomes exposed upon the mitochondrial membrane during apoptosis. As a general standard for identifying that apoptosis had occurred, the cells were assessed for the presence of DNA-laddering on agar gel electrophoresis and by demonstration of characteristic cell morphology. Results were as follows: Fluorescein Isothiocyanate (FITC)-labelled Annexin V/Propidium iodide flow cytometry appeared to be the most sensitive, the most specific and the most user-friendly test for measurement of apoptosis of cells in culture conditions in suspension. The expression of 7A6 antigen on the mitochondrial membrane appeared to be not specific for apoptotic cell death.     

Flow Cytometry Bioinformatics

Flow cytometry bioinformatics is the application of bioinformatics to flow cytometry data, which involves storing, retrieving, organizing, and analyzing flow cytometry data using extensive computational resources and tools. Flow cytometry bioinformatics requires extensive use of and contributes to the development of techniques from computational statistics and machine learning. Flow cytometry and related methods allow the quantification of multiple independent biomarkers on large numbers of single cells. The rapid growth in the multidimensionality and throughput of flow cytometry data, particularly in the 2000s, has led to the creation of a variety of computational analysis methods, data standards, and public databases for the sharing of results. Computational methods exist to assist in the preprocessing of flow cytometry data, identifying cell populations within it, matching those cell populations across samples, and performing diagnosis and discovery using the results of previous steps. For preprocessing, this includes compensating for spectral overlap, transforming data onto scales conducive to visualization and analysis, assessing data for quality, and normalizing data across samples and experiments. For population identification, tools are available to aid traditional manual identification of populations in two-dimensional scatter plots (gating), to use dimensionality reduction to aid gating, and to find populations automatically in higher dimensional space in a variety of ways. It is also possible to characterize data in more comprehensive ways, such as the density-guided binary space partitioning technique known as probability binning, or by combinatorial gating. Finally, diagnosis using flow cytometry data can be aided by supervised learning techniques, and discovery of new cell types of biological importance by high-throughput statistical methods, as part of pipelines incorporating all of the aforementioned methods. Open standards, data, and software are also key parts of flow cytometry bioinformatics. Data standards include the widely adopted Flow Cytometry Standard (FCS) defining how data from cytometers should be stored, but also several new standards under development by the International Society for Advancement of Cytometry (ISAC) to aid in storing more detailed information about experimental design and analytical steps. Open data is slowly growing with the opening of the CytoBank database in 2010 and FlowRepository in 2012, both of which allow users to freely distribute their data, and the latter of which has been recommended as the preferred repository for MIFlowCyt-compliant data by ISAC. Open software is most widely available in the form of a suite of Bioconductor packages, but is also available for web execution on the GenePattern platform.

This is a “Topic Page” article for PLOS Computational Biology.

     

Honey Bee Hemocyte Profiling by Flow Cytometry

Multiple stress factors in honey bees are causing loss of bee colonies worldwide. Several infectious agents of bees are believed to contribute to this problem. The mechanisms of honey bee immunity are not completely understood, in part due to limited information about the types and abundances of hemocytes that help bees resist disease. Our study utilized flow cytometry and microscopy to examine populations of hemolymph particulates in honey bees. We found bee hemolymph includes permeabilized cells, plasmatocytes, and acellular objects that resemble microparticles, listed in order of increasing abundance. The permeabilized cells and plasmatocytes showed unexpected differences with respect to properties of the plasma membrane and labeling with annexin V. Both permeabilized cells and plasmatocytes failed to show measurable mitochondrial membrane potential by flow cytometry using the JC-1 probe. Our results suggest hemolymph particulate populations are dynamic, revealing significant differences when comparing individual hive members, and when comparing colonies exposed to diverse conditions. Shifts in hemocyte populations in bees likely represent changing conditions or metabolic differences of colony members. A better understanding of hemocyte profiles may provide insight into physiological responses of honey bees to stress factors, some of which may be related to colony failure.     

流式细胞术研究细胞凋亡的方法与技术

细胞发生凋亡时,会伴随着一系列形态学、生物化学及分子生物学性质的变化,包括细胞皱缩,核染色质凝聚,细胞膜通透性改变,Caspases激活,线粒体跨膜电位降低,膜磷酯酰丝氨酸外化,胞质Ca2+浓度升高,DNA片段化及含量变化等特点.应用流式细胞术进行细胞凋亡的研究,对于探讨胚胎发育、衰老以及研究肿瘤的发生、发展和转化等病理生理过程和病毒感染及免疫等具有十分重要的意义.本文就细胞凋亡的特征、基于细胞膜功能的流式细胞术检测方法和基于细胞器功能的流式细胞术检测方法等关键性问题进行了阐述.     

流式细胞术

流式细胞术是一种综合应用光学、机械学、流体力学、电子计算机、细胞生物学、分子免疫学等学科技术,对高速流动的细胞或亚细胞进行快速定量测定和分析的方法。它一秒钟能分析几千个细胞,并同时测定细胞的多个参数,广泛应用于生物医学的许多领域,如测定细胞的特征(形态、膜电位等)和细胞内pH,细胞DNA、蛋白质含量、表面受体、Ca2+等。对生物工程学来说,了解细胞的这些参数尤为重要,因为它们能比用传统技术测得的数据更好地描述细胞群体。从流式细胞仪对细胞多种参数的测定及原理,到它在生物工程学中的应用等方面进行了介绍,并讨论了流式细胞术的局限性和面临的挑战。     

Relationships between DNA Fragmentation, Chromatin Condensation, and Changes in Flow Cytometry Profiles Detected during Apoptosis

The determination of whether a cell dies by apoptosis as opposed to necrosis is usually best made on the basis of distinct structural changes in the chromatin. These changes include extensive condensation of the chromatin and DNA fragmentation. We have shown that the cytotoxic drug bleomycin (BLM) is able to cleave the DNA between the nucleosomes when it enters into the cell. If sufficient amounts of BLM are internalized, the nuclear morphological changes characteristic of apoptosis are detected. In this work, we describe the nuclear changes that occurred after DNA fragmentation as a function of the number of DNA double-strand breaks generated per cell and of the time after their generation. Our results show that DNA fragmentation and degradation of higher-order DNA structure were directly responsible for the nuclear morphological changes associated with apoptosis. During apoptosis reduced fluorescence with respect to the G₀/G₁ cell cycle region (the sub-G₁ region) is often detected if fixed cells from cultures undergoing apoptosis are analyzed by flow cytometry. We demonstrate here that, depending on the extent of the DNA fragmentation and on ulterior changes in chromatin structure, the content of the fluorescent sub-G₁ region can be either soluble pieces of DNA or apoptotic bodies or cells depleted in the DNA content by partial loss of fragmented DNA dissolved in the washing media and/or by the release of apoptotic bodies.     

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.     

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%。     

Plant Chromosome Analysis and Sorting by Flow Cytometry

During the past decade, significant progress has been made in the development of methods for the preparation of plant chromosome suspensions suitable for flow cytometric analysis. In addition to successful classification of chromosomes (flow karyotyping), sorting of single chromosome types with a high degree of purity was reported in several plant species. Sorted chromosomes were used for the establishment of chromosome-specific DNA libraries and for gene mapping. The results confirmed the potential of plant flow cytogenetics and form a solid basis for further progress in this area. This article reviews its current status, analyzes major problems, and assesses future directions.     

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。     

Flow Cytometry of Mitotic Cells

We have developed a procedure using flow cytometric measurement of a mitosis-specific antigen that may be used to count mitotic cells and sort them from nonmitotic cells. The procedure may also be used in conjunction with measurement of cellular DNA content and of bromodeoxyuridine incorporation into cellular DNA to assign cells to the G1/G0, S, G2, or M phase of the cell cycle.     

Optimization of Procedures for Counting Viruses by Flow Cytometry

The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems. However, the methods used in flow cytometric analyses of viruses have not been consistent to date. A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here. The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested. A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined. The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at −80°C. Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 × 10⁻⁵ dilution of commercial stock), incubated for 10 min in the dark at 80°C, and cooled for 5 min prior to analysis. The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least −80°C) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.     

Quantitative Assessment of Mycoplasma Hemadsorption Activity by Flow Cytometry

A number of adherent mycoplasmas have developed highly complex polar structures that are involved in diverse aspects of the biology of these microorganisms and play a key role as virulence factors by promoting adhesion to host cells in the first stages of infection. Attachment activity of mycoplasma cells has been traditionally investigated by determining their hemadsorption ability to red blood cells and it is a distinctive trait widely examined when characterizing the different mycoplasma species. Despite the fact that protocols to qualitatively determine the hemadsorption or hemagglutination of mycoplasmas are straightforward, current methods when investigating hemadsorption at the quantitative level are expensive and poorly reproducible. By using flow cytometry, we have developed a procedure to quantify rapidly and accurately the hemadsorption activity of mycoplasmas in the presence of SYBR Green I, a vital fluorochrome that stains nucleic acids, allowing to resolve erythrocyte and mycoplasma cells by their different size and fluorescence. This method is very reproducible and permits the kinetic analysis of the obtained data and a precise hemadsorption quantification based on standard binding parameters such as the dissociation constant K _d. The procedure we developed could be easily implemented in a standardized assay to test the hemadsorption activity of the growing number of clinical isolates and mutant strains of different mycoplasma species, providing valuable data about the virulence of these microorganisms.     

185例儿童急性白血病流式细胞术免疫分型研究

目的:探讨儿童急性白血病流式细胞术免疫分型的意义。方法:采用流式细胞术三色荧光标记技术和CD45/SSC双参数散点图设门,检测185例儿童急性白血病的免疫表型,对抗原表达情况进行分析。结果:流式细胞术免疫分型和FAB分型的符合率为89.19%。185例儿童急性白血病中,ALL为121例,占AL的65.41%,B-ALL为113例,主要表达B系的CD19(99.12%)、CD22(98.13%)、CD79a(96.19%)、CD10(86.73%)。T-ALL占8例;主要表达CD5(100%)、CD7(100%)、cCD3(100%)、CD8(87.5%)。AML为47例,占25.41%,主要表达CD33(93.62%)、CD15(78.72%)、CD64(76.6%)、MPO(76.6%)、CD13(74.47%)。在B-ALL,AML,T-ALL中,敏感性最高的抗体分别是CD19,CD33,CD5和CD7,特异性最强的抗体分别是CD79a,MPO,cCD3。AMLL为17例,占9.19%,其中B/M为9例,T/B为5例,T/M为3例。My十-ALL为54例,占ALL的44.63%,表达的髓系抗原为CD13、CD15、CD33、CD64。Ly+-AML为18例,占AML的38.30%,表达的淋系抗原为CD19、CD4、CD7。系列非相关抗原CD34的表达率为67.57%,HLA-DR的表达率为85.41%,CD38的表达率为80.59%,TdT的表达率为62.59%。结论:流式细胞术免疫分型在白血病分型中起重要作用,是FAB分型的补充和修正,提高了儿童急性白血病诊断的准确率有必要进一步加强流式细胞术免疫分型的标准化工作。     

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.     

Coupling Bacterial Activity Measurements with Cell Sorting by Flow Cytometry

Abstract A new procedure to investigate the relationship between bacterial cell size and activity at the cellular level has been developed; it is based on the coupling of radioactive labeling of bacterial cells and cell sorting by flow cytometry after SYTO 13 staining. Before sorting, bacterial cells were incubated in the presence of tritiated leucine using a procedure similar to that used for measuring bacterial production by leucine incorporation and then stained with SYTO 13. Subpopulations of bacterial cells were sorted according to their average right-angle light scatter (RALS) and fluorescence. Average RALS was shown to be significantly related to the average biovolume. Experiments were performed on samples collected at different times in a Mediterranean seawater mesocosm enriched with nitrogen and phosphorus. At four sampling times, bacteria were sorted in two subpopulations (cells smaller and larger than 0.25 μm³). The results indicate that, at each sampling time, the growth rate of larger cells was higher than that of smaller cells. In order to confirm this tendency, cell sorting was performed on six subpopulations differing in average biovolume during the mesocosm follow-up. A clear increase of the bacterial growth rates was observed with increasing cell size for the conditions met in this enriched mesocosm. Received: 21 January 1999; Accepted: 12 April 1999     

185例儿童急性白血病流式细胞术免疫分型研究

目的：探讨儿童急性白血病流式细胞术免疫分型的意义。方法：采用流式细胞术三色荧光标记技术和CD45／SSC双参数散点图设门,检测185例儿童急性白血病的免疫表型,对抗原表达情况进行分析。结果：流式细胞术免疫分型和FAB分型的符合率为89．19％。185例儿童急性白血病中,ALL为121例,占AL的65．41％,B—ALL为113例,主要表达B系的CD19（99．12％）、CD22（98．13％）、CD79a（96．19％）、CD10（86．73％）。T—ALL占8例;主要表达CD5（100％）、CD7（100％）、cCD3（100％）、CD8（87．5％）。AML为47例,占25．41％,主要表达CD33（93．62％）、CD15（78．72％）、CD64（76．6％）、MPO（76．6％）、CD13（74．47％）。在B—ALL,AML,T—ALL中,敏感性最高的抗体分别是CD19,CD33,CD5和CD7,特异性最强的抗体分别是CD79a,MPO,cCD3。AMLL为17例,占9．19％,其中B／M为9例,T／B为5例,T／M为3例。My＋-ALL为54例,占ALL的44．63％,表达的髓系抗原为CD13、CD15、CD33、CD64。Ly＋-AML为18例,占AML的38．30％,表达的淋系抗原为CD19、CD4、CD7。系列非相关抗原CD34的表达率为67．57％,HLA—DR的表达率为85．41％,CD38的表达率为80．59％,TdT的表达率为62．59％。结论：流式细胞术免疫分型在白血病分型中起重要作用,是FAB分型的补充和修正,提高了儿童急性白血病诊断的准确率。有必要进一步加强流式细胞术免疫分型的标准化工作。