Cytometry最新文献_第5页

Confocal assay for invasion: use of propidium iodide fluorescence and laser reflectance to quantify the rate of migration of cells through a matrix. 侵袭共聚焦测定:使用碘化丙啶荧光和激光反射来量化细胞通过基质的迁移率。

Cytometry Pub Date : 2000-08-01

U Benbow, K A Orndorff, C E Brinckerhoff, A L Givan

{"title":"Confocal assay for invasion: use of propidium iodide fluorescence and laser reflectance to quantify the rate of migration of cells through a matrix.","authors":"U Benbow, K A Orndorff, C E Brinckerhoff, A L Givan","doi":"","DOIUrl":"","url":null,"abstract":"Background: Most assays used to measure invasion are based on manual counting of the number of cells that have migrated completely through commercial coated filters. We describe here a confocal fluorescence-imaging method that can assess the relative rates of invasion of cells into a matrix.Methods: After being seeded on the matrix and a period of incubation, the cells are fixed and treated with RNase. Propidium iodide is then added to stain the double-stranded DNA. A confocal microscope system is used to obtain high-resolution images of the red propidium iodide fluorescence and laser reflectance from optical sections at increasing depths in the matrix. The section with high laser reflectance marks the top of the matrix.Results: Data were calculated as the total area of red fluorescence above background in each section and were plotted as a percentage of the summed fluorescent areas in all sections.Conclusions: Because the distance into the matrix of the nuclei can be calculated by measuring from the reflective upper surface of the matrix, the method is useful for assessing the rate of cell migration and for comparing the ability of different cells to invade through different matrices under varying conditions.","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"40 4","pages":"253-9"},"PeriodicalIF":0.0,"publicationDate":"2000-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21758887","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Cytometric analysis of high shear-induced platelet microparticles and effect of cytokines on microparticle generation. 高剪切诱导血小板微粒的细胞分析及细胞因子对微粒产生的影响。

Cytometry Pub Date : 2000-07-01

S Nomura, T Nakamura, J Cone, N N Tandon, J Kambayashi

{"title":"Cytometric analysis of high shear-induced platelet microparticles and effect of cytokines on microparticle generation.","authors":"S Nomura, T Nakamura, J Cone, N N Tandon, J Kambayashi","doi":"","DOIUrl":"","url":null,"abstract":"Background: Microparticles released from platelets may play a role in the normal hemostatic response to vascular injury, because they exhibit prothrombinase activity. Microparticles are generated by high shear stress and may be formed in diseased small arteries and arterioles in various clinical settings. However, the surface composition of high shear-induced platelet microparticles is unknown. It was recently shown that some cytokines modulate platelet activation. However, no reports are available concerning the effect of cytokines on high shear-induced platelet aggregation (SIPA) microparticle generation.Materials and methods: Measurement of SIPA was performed with a cone-plate viscometer. The conformational characteristics of high shear (108 dynes/cm(2))-induced platelet microparticles were analyzed by flow cytometry and confocal laser scanning microscopy. Effects of cytokines for high SIPA microparticle generation were also analyzed using flow cytometry.Results: The overall pattern of monoclonal antibody binding in high shear-induced microparticles was almost the same as that in activated platelets under high shear stress. Microparticles exhibited markedly increased Annexin V binding. In fluorescent confocal images, small and fine regions of fluorescence (microparticles) were recognized separate from platelet fluorescence. Thrombopoietin not only induced platelet activation, as demonstrated by CD62P expression, but also increased the number of microparticles. Erythropoietin and interleukin-6 enhanced only microparticle generation.Conclusions: These results suggest that microparticles possessing procoagulant activity are released by platelet activation when levels of certain cytokines increase under high shear stress in various clinical settings.","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"40 3","pages":"173-81"},"PeriodicalIF":0.0,"publicationDate":"2000-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21720848","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Response in shape and size of individual p31 cancer cells to cisplatin and ouabain: a computerized image analysis of cell halo characteristics during continuous perfusion. 单个p31癌细胞对顺铂和瓦巴因的形状和大小的反应:连续灌注期间细胞晕特征的计算机图像分析。

Cytometry Pub Date : 2000-07-01

P Behnam-Motlagh, K Grankvist, R Henriksson, K G Engström

{"title":"Response in shape and size of individual p31 cancer cells to cisplatin and ouabain: a computerized image analysis of cell halo characteristics during continuous perfusion.","authors":"P Behnam-Motlagh, K Grankvist, R Henriksson, K G Engström","doi":"","DOIUrl":"","url":null,"abstract":"Background: Volume regulation is essential for cellular functions, including cell death, such as apoptosis. Flow cytometry is standard for nonadherent cells, such as blood cells. Our aim was to explore image analysis methods to study adherent cancer cells of a solid tumor.Methods: P31 mesothelioma cells were perifused (40 min) and studied by phase-contrast microscopy. A noise reduction of the cell contour was tested to more accurately yield the cell shape factor (SF). The optical halo around the cell was analyzed for information about membrane blebbing.Results: The projected cell area (PCA) slowly increased under control perfusion, the halo outside more than the halo inside. Cisplatin (apoptosis) caused an immediate increase in the PCA-halo outside (5.9 +/- 1.2 %, P < 0.01, 1-5 min) and the SF indicated decreased roundness (P < 0.05). The SF-halo inside became more irregular than the outside, which was different from the control cells. The morphology reflected instant blebbing, and the cell bodies showed fragmentation after about 20 min. Ouabain resulted in only small changes in PCA and SF, significantly different from both control and cisplatin conditions.Conclusions: Image analysis (PCA and SF) on perifused adherent cancer cells may serve as a tool to follow the sensitivity of cancer chemotherapy and to study cell death patterns.","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"40 3","pages":"198-208"},"PeriodicalIF":0.0,"publicationDate":"2000-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21720851","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria. 免疫鉴定活菌的二色和三色荧光流式细胞术分析。

Cytometry Pub Date : 2000-07-01

S Barbesti, S Citterio, M Labra, M D Baroni, M G Neri, S Sgorbati

{"title":"Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria.","authors":"S Barbesti, S Citterio, M Labra, M D Baroni, M G Neri, S Sgorbati","doi":"","DOIUrl":"","url":null,"abstract":"Background: Traditional culture methods well established in the past and still in use are not able to detect the environmental microorganisms that exist in a viable but not culturable state. A number of different fluorescence-based assays have been developed over the past decade to detect and identify viable bacteria in the environment.Methods: We have developed a simple and rapid method for measuring the number and viability of immunolabeled bacteria by means of a two/three color fluorescence flow cytometric analysis. After washing, cultured bacteria in suspension were labeled with a rabbit polyclonal antibody recognizing the wall lipopolysaccharide complex. A secondary biotinylated anti-rabbit polyclonal antibody was added allowing the cells to be labeled with the streptavidin R-phycoerythrin-Cyanine 5 (RPE-Cy5) fluorochrome. Before flow cytometric analysis, bacterial suspensions were stained with SYBR Green I and propidium iodide which stain all of the cells and the non viable ones, respectively.Results and conclusions: With the appropriate filter sets of both Bryte-HS (Bio-Rad, Hercules, CA) and FACScan (Becton Dickinson, San Jose, CA) flow cytometers, the measurement of separated green (SYBR Green I), orange-red (propidium iodide), and far red (RPE-Cy5) fluorescence was possible, allowing the enumeration of viable immunodetected bacteria. The entire protocol is completed in less than 3 h, offering numerous possibilities for rapid and precise analyses in sanitary, industrial, and environmental microbiology.","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"40 3","pages":"214-8"},"PeriodicalIF":0.0,"publicationDate":"2000-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21720853","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Characterization of neurosphere cell phenotypes by flow cytometry. 流式细胞术表征神经球细胞表型。

Cytometry Pub Date : 2000-07-01

R Hulspas, P J Quesenberry

{"title":"Characterization of neurosphere cell phenotypes by flow cytometry.","authors":"R Hulspas, P J Quesenberry","doi":"","DOIUrl":"","url":null,"abstract":"Background: Neural stem cell research regularly utilizes neurosphere cultures as a continuous source of primitive neural cells. Results from current progenitor cell assays show that these cultures contain a low number of neural progenitors. Our goal is to characterize neurosphere cultures and define subpopulations in order to purify neural progenitor cells.Methods: Cells from embryonic mouse neurosphere cultures were stained with Hoechst 33342 and analyzed by flow cytometry. Subpopulations were sorted based on their relative fluorescence intensity in the blue and red regions of the spectrum. Individual sorted subpopulations were reanalyzed after 7 days in culture.Results: Neurosphere cultures contain a relatively high number of cells that stain weakly with Hoechst 33342. This subpopulation is present when cultured as an entire batch in the presence of epidermal growth factor (EGF). When cultured separately, this subpopulation gives rise to a neurosphere population with essentially the same characteristics as freshly isolated embryonic mouse brain cells but contains substantially fewer weakly Hoechst-stained cells.Conclusions: Similar to hemopoietic systems, neurosphere cultures contain a subpopulation that can be characterized by a low emission of Hoechst fluorescence. When cultured separately, this subpopulation gives rise to a phenotype similar to freshly isolated, uncultured neural cells.","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"40 3","pages":"245-50"},"PeriodicalIF":0.0,"publicationDate":"2000-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21721479","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Expansions of clonal and oligoclonal T cells in B-cell chronic lymphocytic leukemia are primarily restricted to the CD3(+)CD8(+) T-cell population. b细胞慢性淋巴细胞白血病中克隆和寡克隆T细胞的扩增主要局限于CD3(+)CD8(+) T细胞群。

Cytometry Pub Date : 2000-06-15

C L Goolsby, M Kuchnio, W G Finn, L Peterson

{"title":"Expansions of clonal and oligoclonal T cells in B-cell chronic lymphocytic leukemia are primarily restricted to the CD3(+)CD8(+) T-cell population.","authors":"C L Goolsby, M Kuchnio, W G Finn, L Peterson","doi":"","DOIUrl":"","url":null,"abstract":"B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of mature-appearing clonal B cells exhibiting coexpression of CD5 and CD23. In addition to the accumulation of neoplastic B cells, numerous T-cell abnormalities also occur in B-CLL patients. In this study, the presence, and distribution within the T-cell subsets, of clonal/oligoclonal T cells was studied. Multicolor flow cytometric techniques were employed using combinations of anti-CD3, anti-CD4, and anti-CD8 antibodies coupled with antibodies specific for V(alpha) and V(beta) T-cell receptor (TCR) epitopes. Molecular studies of TCR gene sequences were done to confirm the presence of clonal/oligoclonal T-cell populations. In the flow cytometric studies, examination of V(alpha)/V(beta)expression found evidence of clonal/oligoclonal expansion in 9 of 19 patients studied. In eight of the nine patients, the expansions were restricted to the CD3(+)CD8(+) cell population. Molecular analyses were performed in 16 patients, 12 of whom showed a clonal or oligoclonal pattern. Of the four patients who were negative in the molecular analyses, all demonstrated flow cytometric evidence of clonal/oligoclonal expansions. Thus, when the flow cytometric and molecular analyses were considered together, all 16 patients for whom parallel analyses were done showed evidence of clonal/oligoclonal expansions. These results confirm previous work demonstrating that the majority of B-CLL patients harbor clonal/oligoclonal expansions within the T-cell population. Additionally, based on the relative numbers of cells expressing specific V(alpha) or V(beta)epitopes, these results show that these expansions occur primarily within the CD3(+)CD8(+) T-cell population.","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"42 3","pages":"188-95"},"PeriodicalIF":0.0,"publicationDate":"2000-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21705169","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A rapid measurement of apoptosis-associated light scatter changes using a hematology analyzer. 使用血液学分析仪快速测量细胞凋亡相关的光散射变化。

Cytometry Pub Date : 2000-06-15

T Lertworasirikul, A Bunyaratvej

引用次数: 0

Multisite comparison of methods for the quantitation of the surface expression of CD38 on CD8(+) T lymphocytes. The ACTG Advanced Flow Cytometry Focus Group. CD38在CD8(+) T淋巴细胞表面表达定量方法的多位点比较ACTG高级流式细胞术焦点组。

Cytometry Pub Date : 2000-06-15

J L Schmitz, M A Czerniewski, M Edinger, S Plaeger, R Gelman, C L Wilkening, J A Zawadzki, S B Wormsley

{"title":"Multisite comparison of methods for the quantitation of the surface expression of CD38 on CD8(+) T lymphocytes. The ACTG Advanced Flow Cytometry Focus Group.","authors":"J L Schmitz, M A Czerniewski, M Edinger, S Plaeger, R Gelman, C L Wilkening, J A Zawadzki, S B Wormsley","doi":"","DOIUrl":"","url":null,"abstract":"We evaluated the effect of specimen processing variations and quantitation methods on quantitative determination of CD38 expression on CD8 T lymphocytes. Neither lysing reagent (ammonium chloride versus BD FACSlyse), fixation (paraformaldehyde versus no final fixation step), nor acquisition delay (acquisition within 6 h after fixation versus 24 h after fixation) had a significant effect on CD38 relative fluorescent intensity or CD38 quantitative estimates (RFI or antibodies bound per cell). The only significant difference in fluorescent intensity and CD38 antibodies bound per cell (ABC) was encountered when whole blood was held for 24 h prior to staining and fixation and then acquired after another 24-h hold. However, for all sample processing methods above, the CD4 biologic calibrator and QuantiBRITE bead methods gave significantly different estimates of CD38 intensity. In many cases, however, these differences are relatively small and were more pronounced in certain laboratories. We conclude that there is some flexibility in sample processing methods for quantitative CD38 determination; however, it is preferable for a laboratory to employ one method of fluorescence quantitation calculation consistently because small differences are detected between different methods. Cytometry (Comm. Clin. Cytometry) 42:174-179, 2000.","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"42 3","pages":"174-9"},"PeriodicalIF":0.0,"publicationDate":"2000-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21705167","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Relationship between cytogenetic aberrations by CGH coupled with tissue microdissection and DNA ploidy by laser scanning cytometry in head and neck squamous cell carcinoma. CGH联合组织显微解剖细胞遗传学畸变与激光扫描细胞术检测DNA倍体的关系。

Cytometry Pub Date : 2000-06-01

Y Hashimoto, A Oga, K Okami, Y Imate, Y Yamashita, K Sasaki

引用次数: 0

Simultaneous flow cytometric analyses of enhanced green and yellow fluorescent proteins and cell surface antigens in doubly transduced immature hematopoietic cell populations. 双导未成熟造血细胞群中增强的绿色和黄色荧光蛋白和细胞表面抗原的同时流式细胞术分析。

Cytometry Pub Date : 2000-06-01

R A Stull, W C Hyun, M G Pallavicini

{"title":"Simultaneous flow cytometric analyses of enhanced green and yellow fluorescent proteins and cell surface antigens in doubly transduced immature hematopoietic cell populations.","authors":"R A Stull, W C Hyun, M G Pallavicini","doi":"","DOIUrl":"","url":null,"abstract":"Background: Cell transduction with multiple genes offers opportunities to investigate specific gene interactions on cell function. Detection of multiple transduced genes in hematopoietic cells requires strategies to combine measurements of gene expression with phenotypic cell discriminants. We describe simultaneous flow cytometric detection of two green fluorescent protein (GFP) variants in immunophenotypically defined human hematopoietic subpopulations using only a minor physical adjustment to a standard FACSCalibur.Methods: The accuracy and sensitivity of enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP) detection in mixtures of transduced and nontransduced PG13 packaging cells were evaluated by flow cytometry. Retroviral vectors encoding EGFP or EYFP were used to transduce CD34(+) hematopoietic cells derived from umbilical cord blood. The transduction efficiency into subpopulations of hematopoietic cells was measured using multivariate flow cytometry.Results: A bicistronic retroviral vector containing the EGFP and puromycin N-acetyltransferase (pac) genes afforded brighter EGFP signals in transduced cells than a retroviral vector encoding a pac-EGFP fusion protein. The sensitivity of detecting EGFP and EYFP-expressing cells among a background of nonexpressing cells was 0.01% and 0.05%, respectively. EGFP or EYFP was expressed in up to 95% of CD34(+) DR(-) or CD34(+) 38(-) subpopulations in cord blood 48 h posttransduction. Simultaneous transduction with EGFP and EYFP viral supernatants (1:1 mixture) led to coexpression of both GFP variants in 15% of CD34(+) DR(-) and 20% of CD34(+) 38(-) cells.Conclusions: These results demonstrate simultaneous detection of EGFP and EYFP in immunophenotypically discriminated human hematopoietic cells. This technique will be useful to quantify transduction of multiple retroviral constructs in discriminated subpopulations.","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"40 2","pages":"126-34"},"PeriodicalIF":0.0,"publicationDate":"2000-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21652732","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0