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The article presents basic principles of flow cytometry (FCM), which is a highly advanced technology based on measuring physical and chemical parameters such as: light scatter at different angles and fluorescence. The essential principle of flow cytometry is identifying cell surfaces and intracellular antigens using specific monoclonal antibodies conjugated with fluorochromes. Cells labeled with fluorochromes pass through the flow chamber and, when exposed to the laser beam, they emit and scatter light, causing alterations in the intensity of the light. Light scatter and fluorescence is measured at the same moment. Light signals are converted into digital signals by a photomultiplier system and analyzed by computer. Flow cytometer and computer software provide multiparameter qualitative and quantitative analysis of single cells in a quick, precise and repeatable manner.
The number of ways in which flow cytometry may be applied in veterinary sciences is continually increasing, especially in the fields of immunology, oncology, pharmacology and microbiology. White blood cell analysis by flow cytometry, and in particular, the study of leukocyte antigens, phagocytosis and intracellular killing, is being applied to an ever growing number of animal species. Moreover, flow cytometry enables red blood cell analysis, including reticulocyte count and maturity, identification of anti-erythrocyte antibodies and intraerythrocyte parasites detection. Through flow cytometry it is possible to make a speedy and precise diagnosis of neoplasms. In diseases of the haematopoietic system, such as leukemia and lymphomas this method allows the cell line which has become malignant to be precisely established. When appropriate procedures are used, solid tissues such as tumors can be investigated, and if there is no direct access to tumors, it is possible to study cells from secondary lesions - e.g. pleural fluid or peritoneal fluid. Flow cytometry can provide information about sperm parameters, such as sperm cell chromatin structures or differentiations between X and Y bearing sperm cells. The flow cytometry method enables a wide range of diseases to be diagnosed by studying apoptosis, i.e. programmed cell death. When adequate, modified procedures are applied, it can be utilized in diagnosing bacterial and viral infections and it is the only method which makes it possible to determine cytokines produced by different cell populations, and cytokine sets produced by a single cell.
Przy użyciu cytometru obserwowano zmianę stopnia agregacji szczepów Enterococcus faecalis aph2"(+) pod wpływem szczepów aph2"(-), odpowiednio o niskiej i wysokiej częstości nabywania tego genu oraz odmienne efekty oddziaływań pomiędzy szczepami aph2"(+) i aph2"(-)
The purpose of the study was to attempt the application of flow cytometry to evaluate the sensitivity of various cell lines to EAV infections, according to their type and passage number. Monolayers of RK13, Vero, BHK-21 and MDBK cells were infected with reference EAV strain Bucyrus. First of all the susceptibility of each cell line to different titers of the virus was tested. The second step was to establish a time course kinetic of viral infection. Sequentially, starting from 2 to 72 hours post infection the cells were fixed, permeabilized and stained with FITC monoclonal EAV-specific antibodies. The analysis carried out in Coulter Epics XL (Beckman Coulter) flow cytometer considered the percentage of EAV infected cells determined in a time range by gating FITC/Count histograms. Significant differences in the sensitivity to EAV infection in particular cell lines were found. After 24 h. p.i. most RK13 cells infected with Bucyrus strain showed signs of infection in the titer of 10 TCID₅₀, whilst in Vero and BHK-21 a similar histogram was not obtained until 100 and 1000 TCID₅₀ respectively. As early as 10 and 12 h post inoculation significant level of infected cells (37.9 and 18.6%) were detected in line RK13 passage 36 and 11 respectively. Cell line Vero passage 115 indicated a higher sensitivity to EAV infection comparing to Vero passage 153. More than 70% of cells from that line were EAV infected after 24 hours post inoculation. In Vero passage 153 the infected cells (15.4%) were not detected until 36 h.p.i. The presence of infected cells was found also after 36 h post inoculation in BHK-21 cell line passage 10. The obtained results indicate that differences in cell line susceptibility to EAV infection depend on their type and decrease with their passage.
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