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Sperm chromatin condensation as an in vivo fertility biomarker in bulls:a flow cytometry approach

Journal of Animal Science and Biotechnology 2022年第2期13卷 418-429页

作者： Marc Llavanera Jordi Ribas-Maynou Ariadna Delgado-Bermúdez Sandra Recuero Rodrigo Muiño Carlos OHidalgo Carolina Tamargo Sergi Bonet Yentel Mateo-Otero Marc Yeste Biotechnology of Animal and Human Reproduction(TechnoSperm) Faculty of SciencesUniversity of GironaC/Maria Aurèlia Campany69ES-17003 GironaSpain Department of Biology Unit of Cell BiologyFaculty of SciencesUniversity of GironaES-17003 GironaSpain Department of Animal Pathology Faculty of Veterinary MedicineUniversity of Santiago de CompostelaES-15705 LugoSpain Department of Animal Selection and Reproduction The Regional Agri-Food Research and Development Service of Asturias(SERIDA)E-33394 GijónSpain

Background:Genetic selection in cattle has been directed to increase milk ***,coupled to the fact that the vast majority of bovine artificial inseminations(AI)are performed using cryopreserved sperm,have led to a reduction of fertility rates over the ***,seeking sensitive and specific sperm biomarkers able to predict fertility rates is of vital importance to improve cattle reproductive *** humans,sperm chromatin condensation evaluated through chromomycin A3(CMA3)has recently been purported to be a powerful biomarker for sperm functional status and male *** objectives of the present study were:a)to set up a flow cytometry method for simultaneously evaluating chromatin condensation and sperm viability,and b)to test whether this parameter could be used as a predictor of in vivo fertility in *** study included pools of three independent cryopreserved ejaculates per bull from 25 Holstein *** outcomes of each sire were determined by non-return rates,which were used to classify bulls into two groups(highly fertile and subfertile).Results:Chromatin condensation status of bovine sperm was evaluated through the combination of CMA3 and Yo-Pro-1 staining and flow *** quality parameters(morphology,viability,total and progressive motility)were also *** correlation coefficients and ROC curves were calculated to assess their capacity to predict in vivo *** morphology,viability and total motility presented an area under the ROC curve(AUC)of 0.54,0.64 and 0.68,respectively(P>0.05),and thus were not able to discriminate between fertile and subfertile ***,while the percentage of progressively motile sperm showed a significant predictive value,with an AUC of 0.73(P=0.05),CMA3/Yo-Pro-1 staining even depicted superior results for the prediction of in vivo fertility in ***,the percentage of viable sperm with poor chromatin condensation showed better accura

关键词： Bull Chromatin Chromomycin A3 Condensation Fertility flow cytometry Sperm

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Quantifying and sorting of gold nanoparticle dimers from complex reaction mixtures using flow cytometry

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Nano Research 2016年第10期9卷 3093-3098页

作者： Jens B. Simonsen Nini E. A. Reeler Anna Fossum Knud A. Lerstrup Bo W. Laursen Kasper Nergaard Universitetsparken 5 Nano-Science Center & Department of Chemistry University of Copenhagen Copenhagen DK-2100 Denmark Niels Jensens Vej 2 Sino-Danish Centre for Education and Research (SDC) Aarhus DK-8000 Denmark Ole Maaloes Vej 5 Biotech Research and Innovation Centre (BRIC) University of Copenhagen Copenhagen DK-2200 Denmark

The formation, characterization, and purification of well-defined stoichiometric clusters of metallic nanoparticles, particularly in the form of dimers or trimers, are important and formidable challenges in nanoscience. Here we show that flow cytometry can be used as a high-throughput method to determine the relative distribution of oligomeric clusters of molecularly linked gold nano- particles in bulk solution at the single-particle level with good statistics. This unique information would be near impossible to obtain using traditional characterization techniques. The flow cytometric approach is utilized to provide fast feedback for the synthesis optimization of the complex reaction between citrate-stabilized gold nanoparticles and bi-functional molecular wires with dithiocarbamate anchoring groups. Finally, we demonstrate that flow cytometry can be used to significantly increase the proportion of AuNP dimers from an oligomer-rich polydisperse sample by size-selective sorting.

关键词： flow cytometry characterization sorting nanoparticles gold nanoparticle dimers

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Better understanding of the activated sludge process combining fluorescence-based methods and flow cytometry:A case study

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Journal of Environmental Sciences 2020年第4期32卷 51-58页

作者： Vanesa Benito Javier Etxebarria Felipe Goni-de-Cerio Inigo Gonzalez Pilar Brettes Ana Urkiaga GAIKER Parque TecnológicoEd.20248170ZamudioBizkaiaSpain EDAR Galindo Bilbao Bizkaia Water ConsortiumSestao 48910BizfcaiaSpain

This study aims to demonstrate the validity of fluorescence-based methods,together with flow cytometry,as a complementary tool to conventional physicochemical analyses carried out in wastewater treatment plants(WWTPs),for the control of the currently largely unknown activated sludge *** with SYTO 9,propidium iodide and 5-(and 6)-carboxy-2’,7’-difluorodihydrofluorescein diacetate(carboxy-H2 DFFDA)was used for cell viability and oxidative stress monitoring of the bacterial population forming the activated sludge of a *** the period of research,several unstable periods were detected,where the non-viable bacteria exceeded the 75%of the total bacterial population in the activated sludge,but only in one case the cells with oxidative stress grew to 9%,exceeding the typical values of2%-5%of this *** periods coincided in two cases with high values of total suspended solids(SST)and chemical oxygen demand(COD)in the effluent,and with an excess of ammonia in other case.A correlation between flow cytometric and physicochemical data was found,which enabled to clarify the possible origin of each case of instability in the biological *** experience supports the application of bacterial fluorescence staining,together with flow cytometric analysis,as a simple,rapid and reliable tool for the control and better understanding of the bacteria dynamics in a biological wastewater treatment process.

关键词： Activated sludge Wastewater treatment Cell viability Oxidative stress Fluorescent dyes flow cytometry

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Circulation times of hepatocellular carcinoma cells by in vivo flow cytometry

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Chinese Optics Letters 2010年第10期8卷 953-956页

作者：李延樊志超郭进刘光大谭晓英王成顾正勤魏勋斌 Department of Chemistry Fudan University Institutes of Biomedical Sciences Fudan University Shanghai Medical College Fudan University School of Medical Instrument and Food Engineering University of Shanghai for Science and Technology Department of Urology Xinhua Hospital Shanghai Jiaotong University

Hepatocellular carcinoma （HCC） may metastasize to many organs. The survival rate is almost zero for metastatic HCC patients. Molecular mechanisms of HCC metastasis need to be understood better and new therapies must be developed. We have developed the ＂in vivo microscopy＂ to study the mechanisms that govern liver tumor cells spreading through the microenvironment in vivo. A recently developed ＂in vivo flow cytometer＂ combined with real-time confocal fluorescence imaging is used to assess spreading and the circulation kinetics of liver tumor cells. We measure the depletion kinetics of two related human HCC cell lines, high-metastatic HCCLM3 cells and low-metastatic HepG2 cells, which are from the same origin and obtained by repetitive screenings in mice. More than 60% of the HCCLM3 cells are depleted within the first hour. Interestingly, the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison, less than 40% of the HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes.

关键词： Cell culture flow cytometry Pathology Tumors

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A Sensitive and High-Throughput flow cytometry-Based Assay for Measuring Antibody Neutralization of Human Adenovirus Type 3

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Virologica Sinica 2021年第3期36卷 537-544页

作者： Zhenwei Liu Xingui Tian Wenkuan Liu Yuting Xian Weilue Chen Rong Zhou State Key Laboratory of Respiratory Disease National Clinical Research Center for Respiratory DiseaseGuangzhou Institute of Respiratory HealthThe First Affiliated Hospital of Guangzhou Medical UniversityGuangzhou Medical UniversityGuangzhou 510000China

The assessment of neutralization activity is an important step in the evaluation of neutralizing antibodies (NAbs).The traditional methods for measuring the antibody neutralization of human adenovirus type 3 (HAd V-3) are the microneutralization (MN) assay,which has insufficient sensitivity,and the plaque reduction neutralization test (PRNT),which is not suitable for high-throughput ***,we describe the development of a flow cytometry-based neutralization(FCN) assay for measuring the neutralization of sera,cell culture supernatants,and chimeric antibodies against HAd V-3 on the basis of a recombinant HAd V-3 (r HAd V-3) construct expressing the enhanced green fluorescent protein (EGFP).For flow cytometry-based assays,the optimal cell confluence was determined as 90%,and the virus was titrated using the *** established FCN assay follows the percentage law and an optimal MOI of not less than 5 9 10~(-4)was determined by using a purified chimeric *** addition,comparison of the anti-HAd V-3 NAb titers of 72 human serum samples by the MN and FCN assays,showed that both assays correlated strongly with each *** FCN assay was an improvement over the MN assay because the observation period was reduced from 3 to 1 days and data analysis could be performed objectively and ***,the newly established FCN assay allows measurement of the neutralization activity of chimeric antibodies expressed in cell culture ***,this sensitive and highthroughput FCN assay is a useful alternative to the MN assay for measuring the antibody neutralization of HAd V-3 and for screening anti-HAd V-3 NAbs in cell culture supernatants.

关键词： Sensitive High-throughput flow cytometry Neutralization Adenovirus

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Highly Fluorescent Dye-Doped Silica Nanoparticles Increase flow cytometry Sensitivity for Cancer Cell Monitoring

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Nano Research 2009年第6期2卷 448-461页

作者： M.-Carmen Estévez Meghan B.OʼDonoghue Xiaolan Chen Weihong Tan Center for Research at the Bio/Nano Interface Departments of Chemistry and Department of Physiology and Functional GenomicsShands Cancer Center and UF Genetics InstituteMcKnight Brain InstituteUniversity of FloridaGainesvilleFlorida 32611-7200USA

Early and accurate diagnosis and treatment of cancer depend on rapid,sensitive,and selective detection of tumor *** diagnosis of cancers,especially leukemia,relies on histology and fl ow cytometry using single dye-labeled ***,this combination may not lead to high signal output,which can hinder detection,especially when the probes have relatively weak affi nities or when the receptor is expressed in a low concentration on the target cell *** solve these problems,we have developed a novel method for sensitive and rapid detection of cancer cells using dye-doped silica nanoparticles(NPs)which increases detection sensitivity in fl ow cytometry analyses between 10-and 100-fold compared to standard *** NPs are~60 nm in size and can encapsulate thousands of individual dye molecules within their *** have extensively investigated surface modifi cation strategies in order to make the NPs suitable for selective detection of cancer cells using fl ow *** NPs are functionalized with polyethylene glycol(PEG)to prevent nonspecifi c interactions and with neutravidin to allow universal binding with biotinylated *** virtue of their reliable and selective detection of target cancer cells,these NPs have demonstrated their promising usefulness in conventional fl ow ***,they have shown low background signal,high signal enhancement,and effi cient functionalization,either with antibody-or aptamer-targeting moieties.

关键词： Silica nanoparticle flow cytometry aptamer cell detection polyethylene glycol fl uorophore

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Detection of lntraceUular Proteins and Biomarkers Using Modified Silica Nanoparticles and flow cytometry In vitro

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Chemical Research in Chinese Universities 2018年第2期34卷 229-234页

作者： YU Yang MA Wei YU Lina ZHOU Shuyuan ZHU Guangshan SHI Wei Department of Sports and Health Guangzhou Institute of Physical Education Guangzhou 510500 P. R. China Key Laboratory for Molecular Enzymology and Engineering Ministry of Education College of Life Science Jilin Universily Changchun 130012 P. R. China China-Japan Union Hospital Jilin University Changchun 130033 P. R. China Key Laboratory of Oral Medicine Guangzhou Institute of Oral Disease Stomatology Hospital of Guangzhou Medical University Guangzhou 510140 P. R. China State Key Laboratory of Inorganic Synthesis and Preparative Chemistry College of Chemistry Jilin University Changchun 130012 R R. China Key Laboratory of Polyoxometalate Science Ministry of Education Faculty of Chemistry Northeast Normal University Changchun 130024 P. R. China

In this paper, antibody-modified silica nanoparticles were successfully synthesized, and their average di- ameter was （1094-9） nm. These particles were mixed with cell extracts to target a protein, then, an antibody labeled with fluorescein isothiocyanate（FITC） was added to form FITC-labeled nanoparticle complexes and the product was analyzed using flow cytometry. The results confirm that the intracellular proteins and biomarkers were precisely and sensitivity detected by the novel method in vitro. The FITC intensity of Akt antibody-conjugated particles was in- creased ≥ 10-fold compared with that of control samples in MCF-7 cells. Furthermore, it can also simultaneously measure several proteins when modified with different antibodies.

关键词： Silica nanoparticle flow cytometry Intracellular protein Biomarker

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Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

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Acta Oceanologica Sinica 2009年第2期28卷 103-114页

作者： HOU Jianjun LAI Hongyan HUANG Bangqin CHEN Jixin College of Fisheries Huazhong Agricultural University Wuhan 430070 China College of Life Sciences Hubei Normal University Huangshi 435002 China State Key Laboratory of Marine Environmental Science Environmental Science Research Center Xiamen University Xiamen 361005 China Key Laboratory of Freshwater Fish Germplasm Resources and Biotechnology Yangtse River Fisheries Research Institute Jinzhou 434000 China State Key Laboratory of Freshwater Ecology and Biotechnology Institute of Hydrobiology Chinese Academy of Sciences Wuhan 430072 China

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced, and these sequence data were deposited in the GenBank. Eight oligonucleotide probes （DNA probes） were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order： PM18S02 PM28S02 〉 PM28S01 〉PM18S01, and that of the probes specific to T. pulcheUa was TP18S02 TP28S01 〉 TP28S02 〉TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02, TP18S02 and TP28S01, and the protocol, were also useful for the detection of Mgae in natural samples.

关键词： oligonucleotide DNA probes Prorocentrum minimum Takayama pulcheUa fluorescence in situ hybridization flow cytometry

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Specific Detection of Toxigenic Vibrio cholerae Based on in situ PCR in Combination With flow cytometry

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Biomedical and Environmental Sciences 2007年第1期20卷 64-69页

作者： LI ZHU JUN-PENG CAI QING CHEN SHOU-YI YU School of Public Health and Tropical Medicine Southern Medical University Guangzhou 510515 Guangdong China College of Food Science and Bioengineering South China University of Technology Guangzhou 510641 Guangdong China

Objective To develop an in situ PCR in combination with flow cytometry （ISPCR-FCM） for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. Results A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene （ctxAB gene）. Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low （10^5 cells/mL）, while the specificity was high. Conclusion We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.

关键词： Vibrio cholerae Detection technique in situ PCR flow cytometry

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Genome Size of Alexandrium catenella and Gracilariopsis lemaneiformis Estimated by flow cytometry

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Journal of Ocean University of China 2016年第4期15卷 704-710页

作者： DU Qingwei SUI Zhenghong CHANG Lianpeng WEI Huihui LIU Yuan MI Ping SHANG Erlei Zeeshan Niaz QUE Zhou Key Laboratory of Marine Genetics and Breeding of Ministry of Education of China College of Marine Life Sciences Ocean University of China Department of Microbiology Hazara University MansehraKPK

flow cytometry(FCM) technique has been widely applied to estimating the genome size of various higher plants. However, there is few report about its application in algae. In this study, an optimized procedure of FCM was exploited to estimate the genome size of two eukaryotic algae. For analyzing Alexandrium catenella, an important red tide species, the whole cell instead of isolated nucleus was studied, and chicken erythrocytes were used as an internal reference. The genome size of A. catenella was estimated to be 56.48 ± 4.14 Gb(1C), approximately nineteen times larger than that of human genome. For analyzing Gracilariopsis lemaneiformis, an important economical red alga, the purified nucleus was employed, and Arabidopsis thaliana and Chondrus crispus were used as internal references, respectively. The genome size of Gp. lemaneiformis was 97.35 ± 2.58 Mb(1C) and 112.73 ± 14.00 Mb(1C), respectively, depending on the different internal references. The results of this research will promote the related studies on the genomics and evolution of these two species.

关键词： Alexandrium catenella Gracilariopsis lemaneiformis flow cytometry genome size

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