The Becton Dickinson LSRFortessa is a benchtop analyser equipped with three lasers, a 488nm, a 633nm and a 405nm, which allows up to thirteen parameter analysis (two scatter detectors and eleven colour specific detectors). They are situated within The Immunoassay Biomarker Core Laboratory, L7/181. The Flow Cytometry Core Facility has two multiuser benchtop flow cytometers located within the School Of Medicine, Ninewells Hospital. The Handbook-A Guide to Fluorescent Probes and Labeling Technologies.Detection of rare cell populations such as stem cells, progenitors and immunospecific reactive cellsĮxcellent multimedia presentations on the theory and practice of flow cytometry:Ī comprehensive text on the chemistry and use of fluorescent probes:.Cytoplasmic and nuclear protein determination following permeabilization of the cells.Multiple cytokine determination in a single small volume sample.Intracellular protein phosphorylation determination in cell signalling networks.Functional cell assays such as Calcium Release, Phagocytosis, Reactive Oxygen Species Formation and Cytoplasmic Enzyme Reactions.Cell apoptosis / cell death and viability.Cell immunophenotype using fluorochrome labelled monoclonal antibodies.The flow cytometer provides a platform for the rapid collection of data generated in this way, and this information can be collated into readily decipherable graphical representations.įlow cytometry is commonly used to investigate: Additional information about cell phenotype and function may be gathered by an analysis of light emitted at specific frequencies from excited fluorochromes added to the cells. Information about the cell or particle may be deduced from the scattered laser light in the direction of the laser path (Forward Scatter) and at ninety degrees to the incident laser light (Side Scatter). doi: 10.1016/j.ymeth.2007.11.008.In the technique of flow cytometry a population of cells or other particles is passed in single file in a focused stream of flowing fluid through a laser illumination source. Analysis of apoptosis by cytometry using TUNEL assay. Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis. Flow cytometry of caspase-3 activation in preapoptotic leukemic cells. Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA) correlation with DNA fragmentation. Flow cytometry and biochemical analysis of DNA degradation characteristic of two types of cell death. © 2016 by John Wiley & Sons, Inc.ħ-aminoactinomycin D annexin V apoptosis caspase covalent viability probe fluorogenic caspase substrate propidium iodide.Īfanas'ev, V.N. These relatively simple multiparametric assays are powerful techniques for assessing cell death. In this unit, we will describe the four main techniques for analyzing caspase activity in apoptotic cells, combined with annexin V and cell permeability analysis. The multiparametric nature of flow cytometry makes this technology particularly suited to measuring apoptosis. These assays are particularly powerful when combined into multicolor assays determining several apoptotic characteristics simultaneously. Numerous assays have been devised to measure both the earliest and latest steps in the apoptotic process, from the earliest signal-transduction events to the late morphological changes in cell shape and granularity, proteolysis, and chromatin condensation. Flow cytometry has been the method of choice for analyzing apoptosis in suspension cells for more than 25 years. Apoptosis is also the primary mechanism of action of anti-cancer drugs. It has been particularly well characterized in the immune system, with roles ranging from immature immune cell development and selection to down-regulation of the mature immune response. Apoptosis is an important mechanism in cell biology, playing a critical regulatory role in virtually every organ system.
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