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Technique / Cytometry and fluorescence microscopy / Flow cytometry FACS analysis
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Determining cell cycle stages by flow cytometry. (Curr Protoc Cell Biol. 2001 May;Chapter 8:Unit 8.4.) Darzynkiewicz Z, Juan G, Bedner E. New York Medical College, Valhalla, New York, USA. This unit describes assays used to determine the distribution of a population of cells to the different stages of the cell cycle as analyzed by flow cytometry. Staining the DNA with different fluorescent dyes, propidium iodide or DAPI, is one of the most direct ways of staging the cells based on DNA content. These staining procedures can be used with fixed cells or detergent-permeabilized cells. Alternatively, live cells can be stained with the membrane-permeable stain, Hoechst 33242, and sorted on the basis of DNA content for further culture and characterization. When DNA content measurements are coupled with immunostaining for cyclin expression, it is possible to reliably detect even more stages in progression through the cell cycle. Flow cytometry of apoptosis. (Curr Protoc Cell Biol. 2004 Feb;Chapter 18:Unit 18.8.) Pozarowski P, Grabarek J, Darzynkiewicz Z. School of Medicine, Lublin, Poland. Common methods applicable to flow cytometry make it possible to: (1) identify and quantify dead or dying cells, (2) reveal a mode of cell death (apoptosis or necrosis), and (3) study mechanisms involved in cell death. Gross changes in cell morphology and chromatin condensation, which occur during apoptosis, can be detected by analysis with laser light beam scattering. Early events of apoptosis, dissipation of the mitochondrial transmembrane potential and caspase activation, can be detected using either fluorochrome reporter groups or appropriate antibodies. Exposure of phosphatidylserine on the exterior surface of the plasma membrane can be detected by the binding of fluoresceinated annexin V. Another apoptotic event, DNA fragmentation based on DNA content of cells with fractional ("sub-G1") or DNA strand-break labeling, TUNEL; or In Situ End Labeling, ISEL;. Still another hallmark of apoptosis is the activation of tissue transglutaminase (TGase), the enzyme that crosslinks protein and thereby makes them less immunogenic. The major advantage of flow cytometry in these applications is that it provides the possibility of multiparametric measurements of cell attributes. 
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Bioscience news papers
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Last update: 
03-Jul-2008 07:09 am
- Identification and Characterization of Bacillus anthracis Spores by Multi-Parameter Flow Cytometry. Appl Environ Microbiol. 2008 Jun 27;
Authors: Schumacher WC, Storozuk CA, Dutta PK, Phipps AJ - Genetic characterization of human Dsg3-specific B cells isolated by flow cytometry from the peripheral blood of patients with pemphigus vulgaris. J Dermatol Sci. 2008 Jun 27;
Authors: Yamagami J, Takahashi H, Ota T, Amagai M - Incidence, sensitivity, and specificity of leukemia-associated phenotypes in acute myeloid leukemia using specific five-color multiparameter flow cytometry. Am J Clin Pathol. 2008 Jun;129(6):934-45
Authors: Al-Mawali A, Gillis D, Hissaria P, Lewis I - Analysis of cellular DNA content by flow cytometry. Curr Protoc Immunol. 2004 May;Chapter 5:Unit 5.7
Authors: Darzynkiewicz Z, Huang X - Measurement of intracellular ions by flow cytometry. Curr Protoc Immunol. 2004 Dec;Chapter 5:Unit 5.5
Authors: June CH, Moore JS
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