Variation in nuclear DNA content in an ex-type isolate of Penicillium measured by continuous flow microfluorimetry

Abstract The effect of purified enterotoxin produced by Clostridium difficile on Chinese hamster ovary (CHO) cells was examined. In a certain concentration range (0.9–3.6 μ g/ml), purified toxin caused CHO elongation, namely a cytotonic effect, which is similar to a typical morphological change in CHO cells induced by cholera enterotoxin. At a higher concentration, purified enterotoxin had a cytotoxic effect on CHO cells which was neutralized by anti- C. difficile enterotoxin, but not by anti- C. difficile cytotoxin. Thus, enterotoxin had both cytotonic and cytotoxic effects on CHO cells. About 60 and 180 min were required for binding of enterotoxin to CHO cells, and its internalization, respectively, both times being much longer than those for C. difficile cytotoxin.     

Fundamentals of flow cytometry

Flow cytomelers arc instruments that arc used primarily to measure the physical and biochemical characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents, such as nuclei and organelles. Flow cytomcters are investigative tools for a broad range of scientific disciplines because they make measurements on thousands of individual cells/ particles in a matter of seconds. This is a unique advantage relative to other detection instruments that provide bulk particle measurements. Flow cytomety is a complex and highly technical field; therefore, a basic understanding of the technology is essential for all users. The purpose of this article is to provide fundamental information about the instrumentation used for flow cytometry as well as the methods used to analyze and interpret data. This information will provide a foundation to use flow cytometry effectively as a research tool.     

Use of bicistronic vectors in combination with flow cytometry to screen for effective small interfering RNA target sequences

The efficacy and specificity of small interfering RNAs (siRNAs) are largely dependent on the siRNA sequence. Since only empirical strategies are currently available for predicting these parameters, simple and accurate methods for evaluating siRNAs are needed. To simplify such experiments, target genes are often tagged with reporters for easier readout. Here, we used a bicistronic vector expressing a target gene and green fluorescent protein (GFP) to create a system in which the effect of an siRNA sequence was reflected in the GFP expression level. Cells were transduced with the bicistronic vector, expression vectors for siRNA and red fluorescent protein (RFP). Flow cytometric analysis of the transduced cells revealed that siRNAs for the target gene silenced GFP from the bicistronic vector, but did not silence GFP transcribed without the target gene sequence. In addition, the mean fluorescence intensities of GFP on RFP-expressing cells correlated well with the target gene mRNA and protein levels. These results suggest that this flow cytometry-based method enables us to quantitatively evaluate the efficacy and specificity of siRNAs. Because of its simplicity and effectiveness, this method will facilitate the screening of effective siRNA target sequences, even in high-throughput applications.     

High-resolution flow cytometry of nuclear DNA in higher plants

Summary High-resolution flow cytometry of nuclear DNA in higher plants has been performed from chopped plant tissues and plant protoplasts. A preparation and staining procedure with the DNA specific fluorochrome DAPI, successfully employed for precise flow cytometric DNA analysis of animal and human cells has been used in a slightly modified manner for the DNA analysis of plant cell material. High-resolution DNA histograms coefficients of variation about 1–1.5% have been obtained routinely from plant species with different DNA content. Staining of nuclei with DAPI in combination with the protein fluorochrome sulforhodamine 101 allows bi-parametric analysis of nuclear DNA and protein. The described simple and precise method might be very promising for the analysis of DNA in basic and applied cytogenetic investigations of plant cell research.Abbreviations CV coefficient of variation - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine 101     

Use of molecular markers and flow cytometry to preserve ancient Annurca apple germplasm

The old Annurca apple cultivar (Malus domestica), particularly appreciated for its peculiar flavor and crispy flesh, was studied in order to preserve its ancient germplasm. Twelve clones of Annurca were analyzed using random amplified polymorphic DNA (RAPD) and simple-sequence repeat (SSR) markers. Two out of 30 RAPD primers and nine out of ten SSR primers were able to discriminate all the clones analyzed. Data were confirmed by measuring DNA content using flow cytometry. The results provide a good procedure to improve germplasm field management, in order to removing redundant material in the Annurca collection. This represents an efficient way to create a data bank in order to preserve the genetic variability of the Annurca cultivar. M. Iannaccone, D. Palumbo and I. Ventimiglia contributed equally to this work     

A rapid single step staining technique for DNA analysis by flow microfluorimetry

A new procedure is reported for the staining of DNA, for flow microfluorimetry. It allows the production of stained cell nuclei in a single step by incorporating the DNA stain with a solution of the nonionic detergent Triton-X-100. This method has been found to be applicable to all DNA fluorochromes tested (ethidium bromide, propidium iodide, mithramycin, DAPI, Hoechst 33342). DNA histograms obtained in this way are comparable to those using conventional staining techniques, e.g., ethanol fixation followed by staining. Using this procedure the DNA content distribution of solid tissue or cells from suspension or monolayer cultures can be generated in less than 5 min.     

应用流式细胞术测定17种中国野生蔷薇核DNA含量

以17种中国野生蔷薇为试材,采用改良的LB 01裂解液,以4种不同的标准植物——大豆（Glycine max Merr.‘Polanka’）、绿豆（Vigna radiata（L.） Wilczek）、番茄（Lycopersicon esculentum Miller）和玉米（Zea mays L.）为外标,以二倍体材料丽江蔷薇（Rosa×lichiangensis Yü et Ku）为内部参照,利用流式细胞术对其核DNA含量及染色体倍性进行检测,并采用常规染色体压片法验证倍性准确性。本研究首次检测了3个二倍体种——商城蔷薇（Rosa shangchengensis T.C.Ku）、广东蔷薇（Rosa kwangtungensis Yü et Tsai）和无刺刺梨（Rosa roxburghii f.inermis S.D.Shi）,1个三倍体种——伞房蔷薇（Rosa corymbulosa Rolfe）和1个四倍体种——弯刺蔷薇（Rosa beggeriana Schrenk）的核DNA含量及基因组大小。结果表明,流式细胞术检测结果与常规染色体压片法结果一致,可对中国野生蔷薇的倍性研究进行补充。本研究结果可丰富中国蔷薇属植物的细胞遗传学背景资料并为繁育新品种提供理论依据。     

Quantification of nuclear DNA and G-C content in marine macroalgae by flow cytometry of isolated nuclei

Summary The amounts of nuclear DNA in ten species of seaweeds belonging to the Rhodophyceae, Phaeophyceae, and Chlorophyceae were determined by flow cytometric analysis of nuclei isolated from protoplasts. Genome size was determined from the fluorescence of the nuclei stained with ethidium bromide. The size of the nuclear genome ranged from 0.13 pg per cell in the 1 C population ofUlva rigida to 3.40 pg per cell in the 2 C population ofSphacelaria sp. GC% analysis was based on staining with either Hoechst 33342 or mithramycin A, two fluorochromes specific for the bases A-T and G-C, respectively. Two models were used for the estimation of the proportion of guanine plus cytosine in the nuclear genome. The first one was based on the linear relationships mithramycin A fluorescence/G-C content and ethidium bromide fluorescence/total DNA content. The second model, based on the curvilinear relationships Hoechst 33342 fluorescence/A-T content and mithramycin A fluorescence/G-C content, resulted in comparatively more homogenous and consistent data and appears more accurate. Comparison with previous reports from other methods for the physical investigation of nuclear genomes shows that flow cytometry of nuclei isolated from protoplasts is an accurate, convenient and robust technique to assay for genome sizes and base pair composition in marine macroalgae.Abbreviations A-T nucleic bases adenine and thymine - CRBC chicken red blood cell - FALS forward-angle light scatter - G-C nucleic bases guanine and cytosine - SEIM sorbitol enzymatic incubation medium - SWIM sea water incubation medium - Tm thermal denaturation temperature of DNA     

流式细胞术计数细菌的研究

目的探讨用流式细胞术进行细菌计数。方法检测被链球菌α-34抑止而死亡的化脓性链球菌32309-2数量。结果实验管MFI值明显比对照管强,实验管本身的MFI强度随着时间的增加而增强,而对照管则基本上变化不是很大;到24h后,对照管MFI已达1．01,而对照管还是维持在0．4左右,两者T检验P〈0．01。结论用PI染色,可用流式细胞术检测死亡细菌。结果可靠,用时短,极大地简化了实验步骤。     

Real-time flow cytometry analysis of permeability transition in isolated mitochondria

Mitochondrial membrane permeabilization (MMP) is a key event in necrotic and (intrinsic) apoptotic processes. MMP is controlled by a few major rate-limiting events, one of which is opening of the permeability transition pore (PTP). Here we develop a flow cytometry (FC)-based approach to screen and study inducers and blockers of MMP in isolated mitochondria. Fixed-time and real-time FC permits to co-evaluate and order modifications of mitochondrial size, structure and inner membrane (IM) electrochemical potential (DeltaPsi(m)) during MMP. Calcium, a major PTP opener, and alamethicin, a PTP-independent MMP inducer, trigger significant mitochondrial forward scatter (FSC) increase and side scatter (SSC) decrease, correlating with spectrophotometrically detected swelling. FC-based fluorescence detection of the DeltaPsi(m)-sensitive cationic lipophilic dye JC-1 permits to detect DeltaPsi(m) variations induced by PTP openers or specific inducers of inner MMP such as carbonylcyanide m-chlorophenylhydrazone (mClCCP). These simple, highly sensitive and quantitative FC-based methods will be pertinent to evaluate compounds for their ability to control MMP.     

新鲜肺癌组织的DNA 含量分析

目的：研究新鲜肺癌组织的DNA含量。方法：采用流式细胞术对30例新鲜肺癌组织和5例正常对照组组织制成的单细胞悬液进行了DNA含量分析。结果：肺癌组G0／G1、S、G2／M各时相比率和细胞增殖指数以及DNA指数与对照组存在显著性差异（P〈0．01）。结论：肺病变组织细胞DNA的流式细胞术分析是判定肺部肿瘤恶性化的敏感指标。     

DNA content of wheat monosomics at interphase estimated by flow cytometry

 Two complete, independently maintained sets of 21 monosomic wheat lines derived from cv. ‘Chinese Spring’ were analyzed for their DNA content at the G1 stage with flow cytometry. The DNA content of individual chromosomes was estimated by subtracting the value of a monosomic line from that of euploid wheat. Our data show that the estimated 2C DNA of individual wheat chromosomes in 21 monosomics at the G1 stage ranges from about 0.58 pg in chromosome 1D to approximately 1.12 pg in chromosome 3A. The A genome (2C=6.15 pg) seems to contain more DNA than the B (2C=6.09 pg) and D (2C=5.05 pg) genomes. Analysis of variance showed significant differences (α=0.01) in DNA content both among homoeologous groups and among genomes. Our estimates of interphase DNA content of wheat chromosomes from monosomic lines were poorly correlated to the chromosome sizes at metaphase (r=0.622, P≤0.01). This poor correlation might be due to differential coiling among chromosomes during cell division, possible bias of fluorochrome binding to heterochromatin, or heterogeneity among monosomic lines. Finally, flow cytometry may aid but cannot replace cytological checks in aneuploid maintenance. Received: 21 January 1997 / Accepted: 23 June 1997     

牛分离精子对其IVF胚胎染色体影响的研究

本研究采用传统的细胞遗传学方法,研究了由流式细胞仪分离的、染色未分离的及作为对照用的未染色未分离的分别来自于3头公牛的精子IVF（in vitro fertilization, IVF)后产生的6~8 d囊胚的染色体异常情况,以确定流式细胞仪分离精子的过程及染色对胚胎染色体异常的影响。结果显示,分离精子、染色未分离精子和未染色未分离精子的胚胎中,染色体组成为异常,即嵌合体的胚胎分别占40.7%（59/145）、35.8%（38/106）和37.0%（37/100）,三者染色体异常的比例无显著差异。胚胎染色体异常的频率在不同公牛之间存在差异（33.0% 比 44.6%）（p<0.05）。本研究的结果证明,染料和分离过程没有影响精子的DNA进而影响胚胎的染色体组成;胚胎染色体异常的频率在不同公牛之间存在差异。     

用流式细胞仪测定扇贝血细胞吞噬活性

采用流式细胞仪技术对栉孔扇贝(Chlamysfarreri)和海湾扇贝(Argopectenirradians)血细胞吞噬活性进行了测定。通过对缓冲体系的筛选和比较,建立了扇贝血细胞吞噬活性的流式细胞仪检测方法,并应用该技术对两种扇贝血细胞的吞噬活性进行了比较研究。结果发现在TBS(0.05mol.L-1Tris-HCl,pH7.4;2%glucose;2%NaCl;20mmol.L-1EDTA)和PBS体系中,扇贝血细胞的吞噬活性几乎完全受到抑制,在CMPBS(0.1mol.L-1PBS,pH7.4;2%NaCl;2%glucose;1.0mmol.L-1CaCl2;0.5mmol.L-1MgCl2)体系中细胞吞噬活性略有升高,而在海水缓冲液中细胞吞噬活性最高,其中海湾扇贝和栉孔扇贝血细胞的吞噬率分别达到了26.73%和19.89%,且海湾扇贝血细胞的吞噬率显著高于栉孔扇贝(P<0.05)。研究结果为进一步探讨扇贝血细胞吞噬功能提供了方法依据。     

Establish a flow cytometric method for quantitative detection of Beclin-1 expression

Flow cytometry is an advanced technology for efficient, rapid, specific and multi-parameter analysis of single cells in various basic research fields including cytobiology, immunology, genetic, hematology and other basic research. Beclin-1 protein is an important indicator in monitoring autophagic activity. However, quantitative flow cytometry had been rarely reported till now to be applied in the detection of Beclin-1 expression. The present study was aimed to establish a flow cytometric method for quantitative detection of Beclin-1 expression by employing the autophagy inhibitor 3-methyladenine as the control. A multi-parameter optimal method for Beclin-1 protein staining is as follows. 2 % bovine serum albumin in phosphate buffered saline was used for sample block. Concentration of primary antibody was 0.004 μg/μL. Samples were incubated at room temperature (25 °C) for 30 min. The prepared samples had better to be detected immediately or to be stored at 4 °C and detected within 6 h, otherwise the samples should be fixed in 1 % paraformaldehyde storing at 4 °C and detected within 3 d. Furthermore, we employed the immunohistochemistry to validate the method in vivo, the results confirmed flow cytometric method. The established flow cytometric analysis for Beclin-1 protein has the advantage of simpleness, speediness, sensitivity and reproducibility.     

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.     

A flow cytometric study of c-erbB-3 expression in breast cancer

In order to assess the specificity of biotinylated anti-c-erbB-3 antibody, screening was performed on a series of tumour cell lines and lymphocytes. Staining was found to be consistent, with good reproducibility. Twenty-nine consecutive breast cancer samples were obtained from women treated with tamoxifen and undergoing elective mastectomy. Twenty-eight invasive ductal carcinomas and 1 DCIS were stained for c-erbB-3 expression: 2 were grade I (Bloom and Richardson), 15 grade II, and 11 grade III tumours, 1 being unclassified; 16 were axillary node positive and 10 node negative; in 2 cases no nodes were sampled. Tumours examined by flow cytometry were stained with cytokeratin FITC antibody and the cytokeratin-positive population gated. Using Mann-Whitney analysis no association was seen between c-erbB-3 expression and Bloom and Richardson grade or axillary node status. In the tumour samples c-erbB-3 expression was found to show an association with EGF-R (P=0.021r ²=0.16), PgR (P=0.02,r ²=0.16), c-myc (P<0.0001,r ²=0.5), c-jun (P=0.001,r ²=0.4) and c-fos (P=0.001,r ²=0.5) but not with c-erbB-2 (P=0.2,r ²=0.06), ER (P=0.4,r ²=0.02) or p53 1801 (P=0.05,r ²=0.2). Expression of c-erbB-3 may not be an independent marker of prognosis, but it is associated with other markers of poor prognosis and early cellular events linked with aberrant growth and differentiation.This study was supported by The North of England Cancer Research Campaign     

Endoreduplication of nuclear DNA in the developing maize endosperm

Multiparametric flow cytometry was used to analyze the development of the endosperm in Zea mays L. during the period from 8 to 20 days after pollination (dap). Nuclear size, DNA content per nucleus, and frequencies of nuclei with varying properties were measured in preparations that included all of the endosperm nuclei of single kernels of the inbred strain Al88. Characteristics of nuclear populations from different kernels on the same ear showed minimal variation. The dynamic changes of non-mitotic cells involved in endosperm development consisted of alternating periods of DNA replication with non-replication. Seven rounds of DNA replication had occurred in some nuclei in the later developmental stages with the rate averaging approximately one round per 24-hour period. Analysis of the DNA levels in the nuclei showed an exact doubling pattern indicating an endoreduplication process, that is, replication of the entire genome during each round. The loosely organized polytenization of the chromatin occurred to varying extents among the nuclei within an endosperm. A weak positive correlation existed between DNA content and size of nuclei suggesting that DNA increases and nuclear growth may not be highly coordinated in this tissue. Increased proportions of the larger nuclei occurred in the later stages of endosperm development. Considering the entire endosperm, the average DNA content per nucleus at the 15-dap peak level was approximately 12.8 C constituting a 2.7-fold overall increase from 8 dap.     

p53 expression measured by flow cytometry. A comparison of three monoclonal antibodies and the relationship with grade and DNA ploidy in breast cancer

The aim of this study was to quantify p53 expression by flow cytometry. A panel of three monoclonal antibodies: NCL-p53-240, NCL-p53-1801 and NCL-p53-DO7, was tested on breast cell lines and primary breast cancers. The relationships between ploidy, tumour grade and p53 expression for each antibody, were examined. Methodology was assessed using a variety of breast cell lines. Staining patterns were confirmed and the quantification technique qualified. Cytokeratin-positive cells from 58 samples obtained from patients with breast cancer were assayed for DNA content and p53 expression. p53 quantification was performed using calibrated fluorescent beads on cytokeratin-positive cells. Bloom and Richardson grading revealed 20 grade I and 38 grade II/III breast cancers. Examination of fluorescence thresholds showed a positive correlation between grade and DO7 (P=0.003) at a level of 8900 molecules, 240 (P=0.005) at a level of 2900 molecules and 1801 (P=0.005) at a level of 1850 molecules. These levels equated with 34% (DO7), 43% (240) and 43% (1801) of the samples being classified as p53-positive. Examination of ploidy revealed 23 diploid and 35 aneuploid breast cancers. Application of p53 threshold levels on diploid and aneuploid tumours showed correlation between aneuploidy and p53 expression for DO7 at a level of 9000 molecules, 240 at a level of 1900 molecules and 1801 at a level of 1800 molecules. These levels equated with 34% (DO7), 52% (240) and 52% (1801) of the samples being classified as p53-positive. We conclude that measurement of p53 by flow cytometry may be of clinical importance by indicating levels of positivity using fluorescence thresholds. p53 expression has been shown to correlate with both grade and ploidy. Flow-cytometric measurement of p53 may be a useful prognostic assay.This study was supported by the North of England Cancer Research Campaign