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.     

Flow Cytometry for the Analysis of α-Dystroglycan Glycosylation in Fibroblasts from Patients with Dystroglycanopathies

α-dystroglycan (α-DG) is a peripheral membrane protein that is an integral component of the dystrophin-glycoprotein complex. In an inherited subset of muscular dystrophies known as dystroglycanopathies, α-DG has reduced glycosylation which results in lower affinity binding to several extracellular matrix proteins including laminins. The glycosylation status of α-DG is normally assessed by the binding of the α-DG antibody IIH6 to a specific glycan epitope on α-DG involved in laminin binding. Immunocytochemistry and immunoblotting are two of the most widely used methods to detect the amount of α-DG glycosylation in muscle. While the interpretation of the presence or absence of the epitope on muscle using these techniques is straightforward, the assessment of a mild defect can be challenging. In this study, flow cytometry was used to compare the amount of IIH6-reactive glycans in fibroblasts from dystroglycanopathy patients with defects in genes known to cause α-DG hypoglycosylation to the amount in fibroblasts from healthy and pathological control subjects. A total of twenty one dystroglycanopathy patient fibroblasts were assessed, as well as fibroblasts from three healthy controls and seven pathological controls. Control fibroblasts have clearly detectable amounts of IIH6-reactive glycans, and there is a significant difference in the amount of this glycosylation, as measured by the mean fluorescence intensity of an antibody recognising the epitope and the percentage of cells positive for the epitope, between these controls and dystroglycanopathy patient fibroblasts (p<0.0001 for both). Our results indicate that the amount of α-DG glycosylation in patient fibroblasts is comparable to that in patient skeletal muscle. This method could complement existing immunohistochemical assays in skeletal muscle as it is quantitative and simple to perform, and could be used when a muscle biopsy is not available. This test could also be used to assess the pathogenicity of variants of unknown significance in genes involved in dystroglycanopathies.     

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%。     

Immortalization of Rat Embryo Fibroblasts by a 3′-Untranslated Region

We have exploited a cross-species expression screen to search for cellular immortalizing activities. A newt blastemal cDNA expression library was transfected into rat embryo fibroblasts and immortal cell lines were selected. This identified a 1-kb cDNA fragment which has a low representation in the cDNA library and is derived from the 3′-UTR of an α-glucosidase-related mRNA. Expression of this sequence in rat embryo fibroblasts has shown that it is active in promoting colony formation and immortalization. It is also able to cooperate with an immortalization-defective deletion mutant of SV40 T antigen, indicating that it can exert its growth-stimulatory activity in the pathway activated by a viral immortalizing oncogene. This is the first example of an immortalizing activity mediated by an RNA sequence, and further analysis of its mechanism should provide new insights into senescence and immortalization.     

Analysis of Coccidian Oocyst Populations by Means of Flow Cytometry

Flow cytometry was employed as a tool to analyze and characterize batches of oocysts from laboratory and field isolates of Eimeria spp. from chickens and to propagate sub-populations of batches of oocysts. Oocyst batches were cleaned of debris by a combination of salt flotation, washing and treatment with dilute sodium hypochlorite (1.5% aqueous). Oocyst size and shape were registered by forward-angle light scatter with the argon laser excitation set at 488 nm at 300 mW. Sub-populations of oocysts were collected by map gating and used for microscopy or for propagation. The profile of particle size was characteristic for each species. Propagation of sub-populations of oocysts of specified sizes resulted in cultures of coccidia that were pure species or nearly pure species. The small size of E. mills caused difficulty in separation from the remaining fine debris. This technique was useful for studying the variation in oocyst size within populations and characterization of field isolates of mixed species. Propagation of pure species from mixed isolates by bit-map gating had the same limitations as micromanipulation because of the overlapping size of Eimeria spp. Chancaerization is further limited by the lack of suitable size/shape standards for flow cytometry-Key words. Cell sorter. Eimeria spp., oocyst shape, oocyst size.     

Isolation of Chromosomes from Picea abies and their Analysis by Flow Cytometry

A high-yield method for preparation of suspensions of intact Norway spruce [Picea abies (L.) Karst.] chromosomes was developed for the first time. To accumulate meristem root tip cells at metaphase, actively growing roots were subjected to subsequent treatments with 0.625 mM hydroxyurea for 18 h and after 8 h recovery in distilled water with 0.05 % (m/v) colchicine for 8 h. These treatments resulted in 50 % metaphase indices. Synchronized root tips were fixed in 2 % formaldehyde for 10 min and chromosomes were released into a lysis buffer by mechanical homogenisation, producing 5 × 10⁵ chromosomes from 50 root tips, at average. The isolated chromosomes were morphologically intact and suitable for flow cytometric analysis. Flow karyotypes obtained after the analysis of DAPI-stained chromosomes indicated a possibility to sort at least three different chromosome types. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of Flow Cytometry Data by Matrix Relevance Learning Vector Quantization

Flow cytometry is a widely used technique for the analysis of cell populations in the study and diagnosis of human diseases. It yields large amounts of high-dimensional data, the analysis of which would clearly benefit from efficient computational approaches aiming at automated diagnosis and decision support. This article presents our analysis of flow cytometry data in the framework of the DREAM6/FlowCAP2 Molecular Classification of Acute Myeloid Leukemia (AML) Challenge, 2011. In the challenge, example data was provided for a set of 179 subjects, comprising healthy donors and 23 cases of AML. The participants were asked to provide predictions with respect to the condition of 180 patients in a test set. We extracted feature vectors from the data in terms of single marker statistics, including characteristic moments, median and interquartile range of the observed values. Subsequently, we applied Generalized Matrix Relevance Learning Vector Quantization (GMLVQ), a machine learning technique which extends standard LVQ by an adaptive distance measure. Our method achieved the best possible performance with respect to the diagnoses of test set patients. The extraction of features from the flow cytometry data is outlined in detail, the machine learning approach is discussed and classification results are presented. In addition, we illustrate how GMLVQ can provide deeper insight into the problem by allowing to infer the relevance of specific markers and features for the diagnosis.     

流式细胞术揭示出枯草芽孢杆菌多态异质性

新近的研究发现,微生物群体异质性现象普遍存在,与微生物群体许多关键功能密切相关.微生物群体中的多种异质性状态需要单细胞水平的分析技术才能被揭示,流式细胞术是获取异质性状态精确分布的重要工具.但微生物细胞尺寸微小、生物分子含量少、常常缺乏特异性试剂等都限制着传统流式细胞技术在微生物研究领域的应用.本论文采用新型的低背景、高灵敏度和高分辨率流式细胞仪,以增强的前向散射光、侧向散射光以及紫外光激发的细菌自发荧光水平这三个无需任何荧光标记就可以检测的信号为参数,首次揭示出不同生长状态的枯草芽孢杆菌具有复杂、动态的异质性状态分布.这一方法鉴定出的枯草芽孢杆菌多种状态及其与生理功能相关的、高度关联的变化,可能对该菌的生理变化规律及其分子机理的认识提供新的机遇.本论文也讨论了这一采用新型高灵敏度、高分辨率流式细胞仪测量非标记细胞参数的方法对于广泛开展各种微生物多态性研究具有巨大潜力.     

Flow Cytometry: Long-Term Storage of Propidium Iodide/Citrate-Stained Material

Single cell suspensions stained by the propidium iodide/hypotonic citrate method for DNA content analysis by flow cytometry can be mixed with an equal amount of 70% alcohol for long-term storage and shipping without introduction of artifacts or loss of fluorescence.     

FC-TRIPLEX Chagas/Leish IgG1: A Multiplexed Flow Cytometry Method for Differential Serological Diagnosis of Chagas Disease and Leishmaniasis

Differential serological diagnosis of Chagas disease and leishmaniasis is difficult owing to cross-reactivity resulting from the fact that the parasites that cause these pathologies share antigenic epitopes. Even with optimized serological assays that use parasite-specific recombinant antigens, inconclusive test results continue to be a problem. Therefore, new serological tests with high sensitivity and specificity are needed. In the present work, we developed and evaluated the performance of a new flow cytometric serological method, referred to as FC-TRIPLEX Chagas/Leish IgG1, for the all-in-one classification of inconclusive tests. The method uses antigens for the detection of visceral leishmaniasis, localized cutaneous leishmaniasis, and Chagas disease and is based on an inverted detuned algorithm for analysis of anti-Trypanosomatidae IgG1 reactivity. First, parasites were label with fluorescein isothiocyanate or Alexa Fluor 647 at various concentrations. Then serum samples were serially diluted, the dilutions were incubated with suspensions of mixed labeled parasites, and flow cytometric measurements were performed to determine percentages of positive fluorescent parasites. Using the new method, we obtained correct results for 76 of 80 analyzed serum samples (95% overall performance), underscoring the outstanding performance of the method. Moreover, we found that the fluorescently labeled parasite suspensions were stable during storage at room temperature, 4°C, and –20°C for 1 year. In addition, two different lots of parasite suspensions showed equivalent antigen recognition; that is, the two lots showed equivalent categorical segregation of anti-Trypanosomatidae IgG1 reactivity at selected serum dilutions. In conclusion, we have developed a sensitive and selective method for differential diagnosis of Chagas disease, visceral leishmaniasis, and localized cutaneous leishmaniasis.     

Analysis of Mopc-315 Plasmacytoma By Elutriation and Flow Cytometry

ABSTRACT Intravenously transplanted murine plasmacytoma MOPC-315 cells were separated from normal spleen cells from a tumour-bearing mouse by elutriation and characterized according to morphology, immunologic properties and clonogenicity. Morphologically, both lymphocytoid and plasmacytoid cells were separable by elutriation. Flow cytometry correlated DNA content and intracytoplasmic IgA content and demonstrated two distinct populations, both in cell cycle, but with markedly different cellular IgA levels. Density gradient separation characterized the lower-density cells with lower IgA content and higher clonogenicity. From these studies a model of cellular differentiation is proposed.     

Repair efficiency of (5′S)-8,5′-cyclo-2′-deoxyguanosine and (5′S)-8,5′-cyclo-2′-deoxyadenosine depends on the complementary base

5′-R and 5′-S diastereoisomers of 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG) containing a base-sugar covalent bond are formed by hydroxyl radicals. R-cdA and S-cdA are repaired by nucleotide excision repair (NER) in mammalian cellular extracts. Here, we have examined seven purified base excision repair enzymes for their ability to repair S-cdG or S-cdA. We could not detect either excision or binding of these enzymes on duplex oligonucleotide substrates containing these lesions. However, both lesions were repaired by HeLa cell extracts. Dual incisions by human NER on a 136-mer duplex generated 24–32 bp fragments. The time course of dual incisions were measured in comparison to cis-anti-B[a]P-N²-dG, an excellent substrate for human NER, which showed that cis-anti-B[a]P-N²-dG was repaired more efficiently than S-cdG, which, in turn, was repaired more efficiently than S-cdA. When NER efficiency of S-cdG with different complementary bases was investigated, the wobble pair S-cdG·dT was excised more efficiently than the S-cdG·dC pair that maintains nearly normal Watson-Crick base pairing. But S-cdG·dA mispair with no hydrogen bonds was excised less efficiently than the S-cdG·dC pair. Similar pattern was noted for S-cdA. The S-cdA·dC mispair was excised much more efficiently than the S-cdA·dT pair, whereas the S-cdA·dA pair was excised less efficiently. This result adds to complexity of human NER, which discriminates the damaged base pairs on the basis of multiple criteria.     

High‐Throughput Quantitation of Intracellular Trafficking and Organelle Disruption by Flow Cytometry

Current methods for the quantitation of membrane protein trafficking rely heavily on microscopy, which has limited quantitative capacity for analyses of cell populations and is cumbersome to perform. Here we describe a simple flow cytometry‐based method that circumvents these limitations. The method utilizes fluorescent pulse‐width measurements as a highly sensitive indicator to monitor the changes in intracellular distributions of a fluorescently labelled molecule in a cell. Pulse‐width analysis enabled us to discriminate cells with target proteins in different intracellular locations including Golgi, lyso‐endosomal network and the plasma membrane, as well as detecting morphological changes in organelles such as Golgi perturbation. The movement of endogenous and exogenous retrograde cargo was tracked from the plasma membrane‐to‐endosomes‐to‐Golgi, by decreasing pulse‐width values. A block in transport upon RNAi‐mediated ablation of transport machinery was readily quantified, demonstrating the versatility of this technique to identify pathway inhibitors. We also showed that pulse‐width can be exploited to sort and recover cells based on different intracellular staining patterns, e.g. early endosomes and Golgi, opening up novel downstream applications. Overall, the method provides new capabilities for viewing membrane transport in thousands of cells per minute, unbiased analysis of the trafficking of cargo, and the potential for rapid screening of inhibitors of trafficking pathways.      

人永生化角朊细胞表达干细胞表型的流式细胞术分析

观察人永生化角朊细胞系HaCaT细胞表达的几种干细胞表型,为以该细胞系构建人组织工程皮肤和进行基因操作提供相应的实验依据。方法:采用无血清培养基对HaCaT细胞进行培养,获取处于对数生长期的细胞进行直接荧光双标法、直接荧光单标法和间接荧光单标法以分别CD49fCD71、角蛋白K19、CD29和CD133,依托流式细胞仪检测上述抗原在HaCaT细胞的表达情况。结果:特异性较高的角朊干细胞表型CD49f D71-和K19 在HacaT细胞的百分率分别为16．6±2．8%和14．94±1．23%。CD29 HaCaT细胞为49．55±6．68%,可能包括了角朊干细胞和短暂扩增细胞。分别反映细胞黏附特性和增殖能力的CD49f HaCaT细胞和CD71 HaCaT细胞各占98．1±0．8%和82．1±3．9%。HaCaT细胞仅表达3．43±0．77%的干细胞表型CD133。结论:人永生化角朊细胞系HaCaT细胞的表型特征表明它具有较高的角朊干细胞和短暂扩增细胞比例以及很强的黏附基底膜特性和增殖能力,提示它可以作为构建人组织工程皮肤种子细胞和基因修饰角朊干细胞的替代物。     

利用流式细胞术评价长期冻存对PBMCs各亚型的影响

目的:利用流式细胞术,检测长期冻存后PBMCs总数及各亚型的变化,评价PBMCs的长期冻存效果。方法:收集志愿者外周血PBMCs,利用流式细胞术分析液氮冻存后PBMCs细胞总数及其亚型的变化。结果:长期冻存后,PBMCs总细胞量和细胞活力无显著改变(P=0.19, P=0.32);T细胞、NK细胞和NKT细胞比例无明显变化,但B细胞比例增多(P0.01),单核细胞比例显著减少(P0.001);低温保存影响活化的T细胞和Tregs细胞数量(P0.05, P0.05),其中初始Tregs和记忆Tregs显著减少(P0.05,P0.01)。结论:PBMCs长期冻存会影响B细胞,单核细胞、活化T细胞和Tregs细胞的活性。     

Control by Nutrients of Growth and Cell Cycle Progression in Budding Yeast, Analyzed by Double-Tag Flow Cytometry

To gain insight on the interrelationships of the cellular environment, the properties of growth, and cell cycle progression, we analyzed the dynamic reactions of individual Saccharomyces cerevisiae cells to changes and manipulations of their surroundings. We used a new flow cytometric approach which allows, in asynchronous growing S. cerevisiae populations, tagging of both the cell age and the cell protein content of cells belonging to the different cell cycle set points. Since the cell protein content is a good estimation of the cell size, it is possible to follow the kinetics of the cell size increase during cell cycle progression. The analysis of the findings obtained indicates that both during a nutritional shift-up (from ethanol to glucose) and following the addition of cyclic AMP (cAMP), two important delays are induced. The preexisting cells that at the moment of the nutritional shift-up were cycling before the Start phase delay their entrance into S phase, while cells that were cycling after Start are delayed in their exit from the cycle. The combined effects of the two delays allow the cellular population that preexisted the shift-up to quickly adjust to the new growth condition. The effects of a nutritional shift-down were also determined.     

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) α and β loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders^1-4. T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the αβTCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the αβTCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC⁵.Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events⁶. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes^7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCRα chain at the DN stage, resulting in premature negative selection. Our lab has developed the HY^cd4 model, in which the transgenic HY TCRα is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice¹⁰.Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HY^cd4 mouse model. While negative selection in HY^cd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.     

An Evaluation of the Effects of Cytokines on Intracellular Oxidative Production in Normal Neutrophils by Flow Cytometry

To evaluate the effects of inflammatory cytokines on oxidative production in normal neutrophils, seven kinds of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), interleukin-2 (IL-2), IL-6, IL-1α IL-1β, and interferon-β (IFN-β) were tested. The intracellular hydrogen peroxide (H₂O₂) in individual cells was determined by flow cytometry. According to the levels of intracellular H₂O₂ enhanced by cytokines, these seven cytokines were classified into three types: (1) prominently effective—GM-CSF; (2) moderately effective—G-CSF, IL-6, and IL-2; (3) weakly or ineffective—IFN-β, IL-1α, and IL-1β. Changes in cell size and cell surface structure after stimulation of those seven cytokines were simultaneously measured by flow cytometry. The most prominently effective cytokine, GM-CSF, initially caused enlargement of cell size and irregularity of the cell surface and subsequently increased H₂O₂ production by neutrophils. In contrast, the weakly or ineffective cytokines, like IL-1β, had no effects on cell size or cell surface. Our study indicates that some kinds of cytokines enhance oxidetive production and cause morphological changes in neutrophils.     

巴西橡胶树栽培种质基因组C值测定和变异分析

为了解巴西橡胶树(Hevea brasiliensis)栽培种质的变异情况,以53份在云南植胶区综合性状表现较好的巴西橡胶树栽培种质为材料,采用流式细胞术测定了基因组C值,并进行了变异分析。结果表明,浅绿色嫩叶是巴西橡胶树流式细胞术测定的最适样品。53份巴西橡胶树栽培种质的细胞核DNA含量和基因组C值存在一定差异,基因组的平均C值是1.531 696×109 bp,最小的是CRTG-272种质(1.465 908×10~9 bp),最大的是CRTG-83种质(1.600 381×10~9 bp),变异系数较小(CV=0.035 5)。53份巴西橡胶树栽培种质中有47份为二倍体,6份为三倍体。在已测定基因组大小的40种大戟科(Euphorbiaceae)植物中,基因组大小变异较大(CV=1.248 6),与"C值悖论"观点相一致。因此,应用流式细胞术能快速、准确地测定巴西橡胶树细胞核DNA含量、基因组C值和染色体倍性。