Cytometry最新文献

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Design and first results of CytoBuoy: a wireless flow cytometer for in situ analysis of marine and fresh waters. CytoBuoy的设计和初步结果:一种用于海洋和淡水原位分析的无线流式细胞仪。
Cytometry Pub Date : 1999-12-01
G B Dubelaar, P L Gerritzen, A E Beeker, R R Jonker, K Tangen
{"title":"Design and first results of CytoBuoy: a wireless flow cytometer for in situ analysis of marine and fresh waters.","authors":"G B Dubelaar,&nbsp;P L Gerritzen,&nbsp;A E Beeker,&nbsp;R R Jonker,&nbsp;K Tangen","doi":"","DOIUrl":"","url":null,"abstract":"<p><strong>Background: </strong>The high costs of microscopical determination and counting of phytoplankton often limit sampling frequencies below an acceptable level for the monitoring of dynamic ecosystems. Although having a limited discrimination power, flow cytometry allows the analysis of large numbers of samples to a level that is sufficient for many basic monitoring jobs. For this purpose, flow cytometers should not be restricted to research laboratories. We report here on the development of an in situ flow cytometer for autonomous operation inside a small moored buoy or on other platforms.</p><p><strong>Methods and results: </strong>Operational specifications served a wide range of applications in the aquatic field. Specific conditions had to be met with respect to the operation platform and autonomy. A small, battery-operated flow cytometer resulted, requiring no external sheath fluid supply. Because it was designed to operate in a buoy, we call it CytoBuoy. Sampling, analysis, and radio transmission of the data proceed automatically at user-defined intervals. A powerful feature is the acquisition and radio transmission of full detector pulse shapes of each particle. This provides valuable morphological information for particles larger than the 5-microm laser focus.</p><p><strong>Conclusions: </strong>CytoBuoy allows on-line in situ particle analysis, estimation of phytoplankton biomass, and discrimination between different phytoplankton groups. This will increase the applicability of flow cytometry in the field of environmental monitoring.</p>","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"37 4","pages":"247-54"},"PeriodicalIF":0.0,"publicationDate":"1999-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21409251","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
Computer-assisted analysis of epiluminescence microscopy images of pigmented skin lesions. 色素皮肤病变的脱光显微镜图像的计算机辅助分析。
Cytometry Pub Date : 1999-12-01
O Debeir, C Decaestecker, J L Pasteels, I Salmon, R Kiss, P Van Ham
{"title":"Computer-assisted analysis of epiluminescence microscopy images of pigmented skin lesions.","authors":"O Debeir,&nbsp;C Decaestecker,&nbsp;J L Pasteels,&nbsp;I Salmon,&nbsp;R Kiss,&nbsp;P Van Ham","doi":"","DOIUrl":"","url":null,"abstract":"<p><strong>Background: </strong>Epiluminescence microscopy (ELM) is a noninvasive clinical tool recently developed for the diagnosis of pigmented skin lesions (PSLs), with the aim of improving melanoma screening strategies. However, the complexity of the ELM grading protocol means that considerable expertise is required for differential diagnosis. In this paper we propose a computer-based tool able to screen ELM images of PSLs in order to aid clinicians in the detection of lesion patterns useful for differential diagnosis.</p><p><strong>Methods: </strong>The method proposed is based on the supervised classification of pixels of digitized ELM images, and leads to the construction of classes of pixels used for image segmentation. This process has two major phases, i.e., a learning phase, where several hundred pixels are used in order to train and validate a classification model, and an application step, which consists of a massive classification of billions of pixels (i.e., the full image) by means of the rules obtained in the first phase.</p><p><strong>Results: </strong>Our results show that the proposed method is suitable for lesion-from-background extraction, for complete image segmentation into several typical diagnostic patterns, and for artifact rejection. Hence, our prototype has the potential to assist in distinguishing lesion patterns which are associated with diagnostic information such as diffuse pigmentation, dark globules (black dots and brown globules), and the gray-blue veil.</p><p><strong>Conclusions: </strong>The system proposed in this paper can be considered as a tool to assist in PSL diagnosis.</p>","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"37 4","pages":"255-66"},"PeriodicalIF":0.0,"publicationDate":"1999-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21409157","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
Flow cytometric chemosensitivity analysis of blasts from patients with acute myeloblastic leukemia and myelodysplastic syndromes: the use of 7AAD with antibodies to CD45 or CD34. 急性髓母细胞白血病和骨髓增生异常综合征患者细胞的流式细胞术化学敏感性分析:使用7AAD与CD45或CD34抗体
Cytometry Pub Date : 1999-12-01
M Pallis, J Syan, N H Russell
{"title":"Flow cytometric chemosensitivity analysis of blasts from patients with acute myeloblastic leukemia and myelodysplastic syndromes: the use of 7AAD with antibodies to CD45 or CD34.","authors":"M Pallis,&nbsp;J Syan,&nbsp;N H Russell","doi":"","DOIUrl":"","url":null,"abstract":"<p><strong>Background: </strong>Flow cytometry is potentially suited to the chemosensitivity analysis of peripheral blood or bone marrow subpopulations in patients with leukaemia and myelodysplastic syndromes.</p><p><strong>Methods: </strong>The use of the fluorescent dye 7-amino-actinomycin (7AAD) on unfixed cells to measure loss of viability at a range of cytosine arabinoside (ara-C) doses was evaluated. A six-tube flow cytometric assay for measuring the sensitivity to ara-C of CD45/side-scatter-gated or of CD34-positive leukemic blasts with 7AAD was established, using fixed stained normal mononuclear cells as an internal standard for quantitation of viable cells following culture.</p><p><strong>Results: </strong>7AAD dose response curves for 10 patients with acute myeloblastic leukemia (AML) showed a wide range of sensitivities at 2.5-5 microM araC (3.7-97%, mean 54% of control cell viability at 2.5 microM and 4.1-94.6 %, mean 27% at 5 microM). Parallel assays for ATP bioluminescence agreed reasonably well with the 7AAD method, r(s) = 0.78. The chemosensitivity of CD45/SSC-gated blast cells at 2.5 microM araC showed no consistent relationship with the ungated cell populations, such that CD45/SSC-gated blast sensitivity of seven samples ranged from 86% more to 38% less than that of the total population. Similarly, the chemosensitivities of the CD34-gated subpopulations ranged from 51% more to 78% less than those of the total populations.</p><p><strong>Conclusions: </strong>These results emphasize the necessity of measuring the chemosensitivity of the population of interest rather than of the sample as a whole in heterogeneous clinical material.</p>","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"37 4","pages":"308-13"},"PeriodicalIF":0.0,"publicationDate":"1999-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21409163","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
Human basophils express CD22 without expression of CD19. 人嗜碱性细胞表达CD22而不表达CD19。
Cytometry Pub Date : 1999-11-01 DOI: 10.1002/(sici)1097-0320(19991101)37:3<178::aid-cyto3>3.3.co;2-q
K Han, Y Kim, J Lee, J Lim, K Y Lee, C S Kang, W I Kim, B K Kim, S I Shim, S M Kim
{"title":"Human basophils express CD22 without expression of CD19.","authors":"K Han,&nbsp;Y Kim,&nbsp;J Lee,&nbsp;J Lim,&nbsp;K Y Lee,&nbsp;C S Kang,&nbsp;W I Kim,&nbsp;B K Kim,&nbsp;S I Shim,&nbsp;S M Kim","doi":"10.1002/(sici)1097-0320(19991101)37:3<178::aid-cyto3>3.3.co;2-q","DOIUrl":"https://doi.org/10.1002/(sici)1097-0320(19991101)37:3<178::aid-cyto3>3.3.co;2-q","url":null,"abstract":"<p><strong>Background: </strong>Even modern automatic cell counters cannot count basophils precisely. Therefore, we need a rapid, accurate, precise, and easy method for counting basophils.</p><p><strong>Methods: </strong>Using flow cytometry, basophils (CD22+/CD19-) and B cells (CD22+/CD19+) were counted. Within a large lymphocyte light scatter gate, % basophils (G%baso) and % B cells (G%B) were determined from the total count. Another method of analysis was to make two regions (R1 for basophils and R2 for B cells) and to determine in those the % basophils (R1%baso) and % B cells (R2%B) without gating. The flow cytometric basophil counts of the blood of 21 normal controls and 43 chronic myelogenous leukemia (CML) patients were compared with manual basophil count (Ma%baso) and basophil count by Coulter electronic cell counter (Hialeah, FL) (Auto%baso). CD22+/CD19- cells were sorted by a FACSCalibur (Becton Dickinson, San Jose, CA).</p><p><strong>Results: </strong>The G%baso of all samples was 4.66 +/- 5.35%, and R1%baso was 4.23 +/- 4.88%, and they were well-correlated (r = 0.996, P < 0.001). The G%B of all samples was 1.55 +/- 1.68%, and R2%B was 1.59 +/- 1.67%, and they were also well-correlated (r = 0.993, P < 0.001). Their correlation was better in normal controls than in CML. G%baso was well-correlated to Ma%baso (r = 0.827) and Auto%baso (r = 0.806), and R1%baso was well-correlated to Ma%baso (r = 0.831) but showed poor correlation to Auto%baso (r = 0.734). Auto%baso revealed the poorest correlation to Ma%baso (r = 0.692). The sorted CD22+/CD19- cells were all basophils (99.48 +/- 0.30%), and they revealed CD13, CD33, and dim CD45 expression, whereas CD3, CD14, CD16, and HLA-DR were not detected on them.</p><p><strong>Conclusions: </strong>We discovered a specific marker combination to identify basophils (CD22+/CD19-), and we suggest that flow cytometric analysis using these markers is an easy, reliable, and accurate method of basophil counting.</p>","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"37 3","pages":"178-83"},"PeriodicalIF":0.0,"publicationDate":"1999-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21381434","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
Orientation of the chromophore dipoles in the TOTO-DNA system. TOTO-DNA体系中发色团偶极子的取向。
Cytometry Pub Date : 1999-11-01 DOI: 10.1002/(sici)1097-0320(19991101)37:3<230::aid-cyto10>3.3.co;2-r
J M Schins, A Agronskaia, B G de Grooth, J Greve
{"title":"Orientation of the chromophore dipoles in the TOTO-DNA system.","authors":"J M Schins,&nbsp;A Agronskaia,&nbsp;B G de Grooth,&nbsp;J Greve","doi":"10.1002/(sici)1097-0320(19991101)37:3<230::aid-cyto10>3.3.co;2-r","DOIUrl":"https://doi.org/10.1002/(sici)1097-0320(19991101)37:3<230::aid-cyto10>3.3.co;2-r","url":null,"abstract":"<p><strong>Background: </strong>Flow cytometry has been applied successfully to the sizing of medium to large-sized DNA molecules, thanks to the excellent staining properties of cyanine chromophores such as TOTO (homodimer of thiazole orange) (Petty et al.: Anal Chem 67:1755-1761, 1995). The hydrodynamic flow, used to focus the sample molecules in a small laser-illuminated volume, is also responsible for their alignment, thereby allowing the determination of the TOTO-dipole orientation with respect to the DNA axis (Agronskaia et al.: Appl Opt 38:714-719, 1999).</p><p><strong>Methods: </strong>We present model calculations of the fluorescence yield of TOTO-stained DNA measured in a flow-cytometric setup with high numerical aperture. The models consider different orientations of the chromophore dipoles.</p><p><strong>Results: </strong>Comparison of measurement and calculation suggests that the absorption dipoles of the TOTO molecule make a mean angle of 61 degrees with the helix axis of the DNA molecule. This mean angle can be the consequence of two binding modes.</p><p><strong>Conclusions: </strong>Our results indicate that any model with a significant contribution of perpendicularly-oriented chromophores fails to reproduce the experimental results.</p>","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"37 3","pages":"230-7"},"PeriodicalIF":0.0,"publicationDate":"1999-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21381967","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
Forum: journal club 论坛:杂志社
Cytometry Pub Date : 1999-10-15
Macey, Carty, Webb, Chapman, Zelmanovic, Okrongly, Rampton, Newland
{"title":"Forum: journal club","authors":"Macey,&nbsp;Carty,&nbsp;Webb,&nbsp;Chapman,&nbsp;Zelmanovic,&nbsp;Okrongly,&nbsp;Rampton,&nbsp;Newland","doi":"","DOIUrl":"","url":null,"abstract":"","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"38 5","pages":"256-7"},"PeriodicalIF":0.0,"publicationDate":"1999-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21379098","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
Modulation of apoptosis by cytokines in B-cell chronic lymphocytic leukemia. 细胞因子对b细胞慢性淋巴细胞白血病细胞凋亡的调节。
Cytometry Pub Date : 1999-10-15 DOI: 10.1002/(sici)1097-0320(19991015)38:5<224::aid-cyto4>3.3.co;2-s
R Castejón, J A Vargas, Y Romero, M Briz, R M Muñoz, A Durántez
{"title":"Modulation of apoptosis by cytokines in B-cell chronic lymphocytic leukemia.","authors":"R Castejón,&nbsp;J A Vargas,&nbsp;Y Romero,&nbsp;M Briz,&nbsp;R M Muñoz,&nbsp;A Durántez","doi":"10.1002/(sici)1097-0320(19991015)38:5<224::aid-cyto4>3.3.co;2-s","DOIUrl":"https://doi.org/10.1002/(sici)1097-0320(19991015)38:5<224::aid-cyto4>3.3.co;2-s","url":null,"abstract":"<p><p>B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the slow and progressive accumulation of monoclonal apparently mature, CD5(+) B lymphocytes. The majority of circulating cells appear to be nondividing, and it has been suggested that a prolonged life span is mainly responsible for the accumulation of the leukemic cells. However, spontaneous programmed cell death by apoptosis occurs when B chronic lymphocytic leukemia cells are cultured in vitro. This may be because of the lack of an unidentified essential cytokine present in vivo. Thus, we investigate interleukin-2 (IL-2), IL-4, IL-6 and IL-10 in vitro effects on apoptosis of B cells from 32 previously untreated patients with B-CLL in initial clinical stages. B cells were isolated from peripheral blood, and apoptosis was measured in these cells immediately after isolation and following incubation in vitro, without and with the different cytokines, for 24 and 48 h. Distribution of cellular DNA content and quantitative analysis of apoptosis were determined by standard propidium iodide staining and flow cytometry. Spontaneous apoptosis occurred in B-CLL cells incubated in vitro in the absence of cytokines. Our results indicate that both IL-2 and IL-4, but not IL-6, inhibit in vitro apoptosis in a large percentage of B-CLL patients. IL-10 increases in vitro apoptotic cell number in stage 0 patients, but not in stage I and II. These data support the hypothesis that IL-2 or IL-4, may be cell survival factors in vivo and that IL-10 might be a candidate for immune therapy of early B-CLL.</p>","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"38 5","pages":"224-30"},"PeriodicalIF":0.0,"publicationDate":"1999-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21379816","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
Use of mean platelet component to measure platelet activation on the ADVIA 120 haematology system. 在ADVIA 120血液学系统中使用平均血小板成分来测量血小板活化。
Cytometry Pub Date : 1999-10-15 DOI: 10.1002/(sici)1097-0320(19991015)38:5<250::aid-cyto8>3.3.co;2-b
M G Macey, E Carty, L Webb, E S Chapman, D Zelmanovic, D Okrongly, D S Rampton, A C Newland
{"title":"Use of mean platelet component to measure platelet activation on the ADVIA 120 haematology system.","authors":"M G Macey,&nbsp;E Carty,&nbsp;L Webb,&nbsp;E S Chapman,&nbsp;D Zelmanovic,&nbsp;D Okrongly,&nbsp;D S Rampton,&nbsp;A C Newland","doi":"10.1002/(sici)1097-0320(19991015)38:5<250::aid-cyto8>3.3.co;2-b","DOIUrl":"https://doi.org/10.1002/(sici)1097-0320(19991015)38:5<250::aid-cyto8>3.3.co;2-b","url":null,"abstract":"<p><p>Platelet activation results in changes in a number of cell surface molecules including an increase in P-Selectin (CD62P) that may be rapidly and conveniently measured by immunofluorescent flow cytometry. The ADVIA 120 (Bayer) is a new system that facilitates more accurate measurement of platelet volume and in addition provides an approximate measure of the mean refractive index (RI) of the platelets reported as mean platelet component (MPC) concentration. We were interested to determine whether changes in MPC might reflect changes in platelet activation status. To investigate this, the platelet CD62P expression, determined by flow cytometry, and change in MPC, measured on the ADVIA 120 system, was first examined in vitro after stimulation of EDTA anticoagulated whole blood with submaximal concentrations of bovine thrombin in the presence or absence of the thromboxane synthase inhibitor, Ridogrel. Thrombin produced a dose-dependent increase in platelet CD62P expression and a decrease in MPC that could be inhibited by Ridogrel at physiological concentrations. In the second set of experiments, blood from 20 normal controls was collected into both EDTA and sodium citrate (SC) anticoagulants. Within 30 min of venesection and again at 3 h post-venesection after storage at room temperature, the platelet MPC and CD62P expression were determined. Platelets in all samples with both anticoagulants showed very low levels of CD62P expression when first analysed. At 3 h there was a small increase in CD62P expression on platelets in whole blood anticoagulated with SC, but a significant (P < 0.001) increase was observed on platelets anti-coagulated with EDTA. A negative correlation was found between the change in MPC of the platelets and the increase in the mean fluorescence intensity (MFI) (r = -0.69, P < 0.001, n = 20) and the percentage (r = -0.72, P < 0.001, n = 20) of CD62P positive platelets at 3 h in blood anticoagulated with EDTA. We conclude that a reduction in MPC as measured by the ADVIA 120 may be used to detect anticoagulant induced, as well as thrombin stimulated, in vitro platelet activation in blood anticoagulated with EDTA. Further, we conclude that platelet activation is negligible for up to 3 h in sodium citrate anticoagulated whole blood.</p>","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"38 5","pages":"250-5"},"PeriodicalIF":0.0,"publicationDate":"1999-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21379097","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
Bivariate analysis of the p53 pathway to evaluate Ad-p53 gene therapy efficacy. 通过双变量分析评价Ad-p53基因治疗的疗效。
Cytometry Pub Date : 1999-10-15 DOI: 10.1002/(sici)1097-0320(19991015)38:5<201::aid-cyto2>3.3.co;2-6
J W Jacobberger, R M Sramkoski, D Zhang, L A Zumstein, L D Doerksen, J A Merritt, S A Wright, K E Shults
{"title":"Bivariate analysis of the p53 pathway to evaluate Ad-p53 gene therapy efficacy.","authors":"J W Jacobberger,&nbsp;R M Sramkoski,&nbsp;D Zhang,&nbsp;L A Zumstein,&nbsp;L D Doerksen,&nbsp;J A Merritt,&nbsp;S A Wright,&nbsp;K E Shults","doi":"10.1002/(sici)1097-0320(19991015)38:5<201::aid-cyto2>3.3.co;2-6","DOIUrl":"https://doi.org/10.1002/(sici)1097-0320(19991015)38:5<201::aid-cyto2>3.3.co;2-6","url":null,"abstract":"<p><strong>Background: </strong>Gene therapy of human tumors with adenovirus vectors presents a clinical research challenge and a potential opportunity in cancer therapy. One of the research challenges is that endpoints like tumor reduction, time to recurrence, and survival do not provide information about whether a potential therapeutic infects the targeted cells or whether the transferred gene functions or induces a cellular response. Therefore, a flow cytometric approach was developed for a wildtype, p53 encoding adenoviral vector (Ad-p53) that provides (1) the relative level of p53 transferred by p53 immunoreactivity, (2) mdm2 immunoreactivity as an assay of p53 activity, and (3) estimates of the percentage of infected cells by dual parameter analysis (p53 versus mdm2).</p><p><strong>Methods: </strong>Three prostate cancer cell lines (PC-3, LNCaP, DU 145) that are null, wild-type, and mutant for p53, respectively, and two ovarian cancer cell lines (PA1, MDAH 2774) that are wild-type and mutant for p53, respectively, were tested for immunoreactivity and lack of cross-reactivity with the monoclonal antibodies, DO-7 (anti-p53) and IF2 (anti-mdm2). Optimal dual staining conditions for a flow cytometric assay employing saturating levels of antibody were developed and tested by infection of PC-3, PA1, and MDAH 2774 with Ad-p53 or a control virus, Ad-luc. Dual staining with DO-7 and propidium iodide was used to determine any biological effect of the transferred gene.</p><p><strong>Results: </strong>Neither DO-7 nor IF2 showed appreciable cross-reactions by Western blot analysis of representative prostate or ovarian cell lines. By flow cytometric titration, DO-7 appears to be a high avidity antibody (saturation staining of 10(6) DU 145 cells with 0.5ug) whereas IF2 appears less so (optimum signal to noise ratio at 1ug/10(6) cells). Infection with Ad-p53 was detected at 6 to 48 hours post infection as a uniform relative increase in p53 levels over background p53 levels. Coincident increases in mdm2 immunoreactivity were also detected. DNA content measurements of PA1 and MDAH 2774 cells indicated that G1 arrest and/or apoptosis occurred subsequent to Ad-p53 infection. p53 and mdm2 levels and DNA content distributions for Ad-luc infected cells were equivalent to uninfected cells.</p><p><strong>Conclusions: </strong>A flow cytometric approach to measure the efficacy of an Ad-p53 gene therapy vector was developed that detects not only the gene transferred but also the activity of the transferred gene product.</p>","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"38 5","pages":"201-13"},"PeriodicalIF":0.0,"publicationDate":"1999-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21379814","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
Measuring the complexity of cell cycle arrest and killing of drugs: kinetics of phase-specific effects induced by taxol. 测量细胞周期阻滞和药物杀伤的复杂性:紫杉醇诱导的相特异性效应动力学。
Cytometry Pub Date : 1999-10-01
G Sena, C Onado, P Cappella, F Montalenti, P Ubezio
{"title":"Measuring the complexity of cell cycle arrest and killing of drugs: kinetics of phase-specific effects induced by taxol.","authors":"G Sena,&nbsp;C Onado,&nbsp;P Cappella,&nbsp;F Montalenti,&nbsp;P Ubezio","doi":"","DOIUrl":"","url":null,"abstract":"<p><strong>Background: </strong>Paclitaxel (Taxol) is known to act mainly in mitosis, interfering with microtubule dynamics, but effects on the other cells cycle phases have been reported also. However, a comparative picture of perturbation and killing in the G(1), S and G(2)M phases after drug treatment is lacking. The approach developed by our group tackles the problem of the complexity of cell cycle effects with the aid of a computer program simulating cell cycle progression and new quantities measuring cell-cycle arrest and death.</p><p><strong>Methods: </strong>The program generates data that were compared with those given by absolute cell counts and by different flow cytometry techniques, enabling us to follow the fate of G(1) and G(2)M blocked cells either re-entering the cycle or dying, distinguishing cytostatic and cytotoxic effects. Apoptosis was analyzed in order to refine the description of cytotoxic effects.</p><p><strong>Results: </strong>We estimated the number of blocked and dead cells after short-term Taxol treatments in a range of concentrations and post-drug incubation times. G(2)M block was immediately active at low concentrations but was reversible, becoming irreversible only at the highest concentrations. G(1)block became active later, allowing cell cycle progression of cells initially in G(1), but was still active 48 h post-treatment, at intermediate concentrations. S-phase delay was detected after 24 h. The death rate was much higher within G(1)than G(2)M blocked cells.</p><p><strong>Conclusions: </strong>Our analysis unraveled the complexity of cell cycle effects of the drug, and revealed the activity of G(1) checkpoint, hidden by a prompter but less cytotoxic G(2)M block.</p>","PeriodicalId":10947,"journal":{"name":"Cytometry","volume":"37 2","pages":"113-24"},"PeriodicalIF":0.0,"publicationDate":"1999-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21349357","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
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