Flow cytometry cells

Telford, W G, King, L E, and Fraker, P J (1991) Evaluation of glucocorticoid-induced DNA fragmentation m mouse thymocytes by flow cytometry Cell Proliferation 24,447-459. 

Coba de la Pena, T. and Sanchez-Moreiras, A. M. 2001. Flow cytometry Cell cycle In Reigosa, M.J. (Ed.), Handbook of Plant Ecophysiology Techniques. Kluwer Academic Publishers, Dordrecht, The Netherlands, 65-80... 

Fig. 4. Combined use of flow cytometry/cell sorting and confocal laser scanning microscopy. TNF-a/ActD treated Hepa-1 cells were stained with CMX Rosamine and then analyzed for mitochondrial membrane potential and NAD(P)H fluorescence (A). Cells in different regions of the cytogram were then sorted, and subsequently stained with the DNA fluorochrome Hoechst 33342. These cells were then examined by CLSM. (B), (C), and (D) show three-dimensional reconstructions of nuclei from cells sorted from healthy, early apoptotic, and late apoptotic populations. Whereas healthy cells show normal round nuclei, early and late apoptotic cells show progressive chromatin condensation/margination and nuclear fragmentation.

Cell staining is evaluated by flow cytometry. Cells can be counterstained with viability marker such as Alexafluor 488-Annexin V added to resuspension buffer and incubating for 5 min at room temperature prior to loading of sample. 

Another example of a population study that uses antibodies is flow cytometry. Isolated cells must be dissociated from tissues or cultures and labeled with fluorescent antibodies specific for a subpopulation of the cells. In flow cytometry, cells pass rapidly past a detector that measures the amount of fluorescence for each cell. The size of cells and the amount of fluorescence can be plotted and analyzed. Even though this method makes use of antibodies, it is a population study because it determines the number of isolated cells bound to an antibody. Flow cytometry identifies different populations of isolated cells, but it cannot show the location of these labeled cell in tissues, which can be done only with immunocytochemistry. 

Flow cytometry (cell sorting, 1972) W first high-throughput screening -> with antibodies follow cellular differentiation separate specific cell types... 

Receptor binding Flow cytometry cell surface staining ... 

Differentiation Flow cytometry, cell surface, and intracellular staining ELISA for secreted proteins... 

Aeschbacher, M. Reinhardt, C. A. Zbinden, G. A rapid cell membrane permeability test using fluorescent dyes and flow cytometry. Cell Biol. Toxicol. 1986, 2, 247-255. 

EIA was originally developed as a histological technique to localize specific ceUular sites using the specificity of an immunological reaction (23). The resulting fluorescent antibodies can be detected in animal tissues at levels as low as 1 /tg/mL of body fluid. Eluorophore-labeled antibodies have also been used widely for flow cytometry appHcations using fluorescein antibodies to cell surface markers to detect and quantify specific cells (24). 

Flow cytometer cell counts are much more precise and more accurate than hemocytometer counts. Hemocytometer cell counts are subject both to distributional (13) and sampling (14—16) errors. The distribution of cells across the surface of a hemocytometer is sensitive to the technique used to charge the hemocytometer, and nonuniform cell distribution causes counting errors. In contrast, flow cytometer counts are free of distributional errors. Statistically, count precision improves as the square root of the number of cells counted increases. Flow cytometer counts usually involve 100 times as many cells per sample as hemocytometer counts. Therefore, flow cytometry sampling imprecision is one-tenth that of hemocytometry. 

Figure 5B. Correlation of right-angle light scatter measured by fluorometry and flow cytometry. The top panel shows flow-cytometric data of side scatter of fixed, stained cells during the time course of stimulation by 1-nM (solid line, solid circles) or 0.01-nH (dashed line, open circle) FLPEP. The bottom panel shows the corresponding right-angle light-scatter data acquired pseudo-simultaneously on live cells in the fluorometer. The flow-cytometric data have been averaged, but the fluorometry data are plotted for both duplicates from one donor. Reproduced with permission from Ref. 27. Copyright 1985 Rockefeller University Press.

There is a single assumption in these measurements--namely that the antibody only quenches free ligand. This has been demonstrated specifically by flow cytometry in experiments which show that there is no quenching of ligand on the cell (3). The kinetic analysis depends on rapid interaction of ligand and antibody, which in these experiments is essentially within the mixing time. 

Advantages. The advantages of flow cytometry are numerous. It is the most sensitive of the techniques, able to detect SI,000 fluoro-phores per cell (0.01 nif FLPEP). There is in addition no required total receptor concentration. It is effective for small numbers of... 

Fig. 3. Flow cytometry analysis. CellQuest software. The fluorescence of 50,000 cells is measured.

J. Porter, C. Edwards, J. A. W. Morgan, and R. W. Pickup, Rapid, automated separation of specific bacteria from lake water and sewage by flow cytometry and cell sorting, Appl. Environ. Microbiol. 59 3327 (1993). 

Rituximab is a monoclonal antibody to the CD20 receptor expressed on the surface of B lymphocytes the presence of the antibody is determined during flow cytometry of the tumor cells. Cell death results from antibody-dependent cellular cytotoxicity. The pharmacokinetics of rituximab are best described by a two-compartment model, with a terminal half-life of 76 hours after the first infusion and a terminal half-life of 205 hours after the fourth dose.36 Rituximab has shown clinical activity in the treatment of B-cell lymphomas that are CD20+. Side effects include hypersensitivity reactions, hypotension, fevers, chills, rash, headache, and mild nausea and vomiting. 

Flow cytometry A method of measuring the number of cells in a sample, the percentage of five cells in a sample, and certain characteristics of cells, such as size, shape, and the presence of tumor markers on the cell surface. The cells are stained with a light-sensitive dye, placed in a fluid, and passed in a stream before a laser or other type of light. The measurements are based on how the light-sensitive dye reacts to the light. 

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