• Cell Proliferation, Cell Death, Necrosis & Apoptosis

    Posted on Nov 15, 2018

    For most studies, cell growth is measured by a homogeneous, vital dye method in which one of several choices of dye is added to cells in a 96 well plate at the conclusion of the study or at time point intervals, and read directly in a plate reader. The dye is enzymatically changed in healthy cells so that development of color or fluorescence is measured using a different wavelength than the unaltered dye. The effects of adding a growth factor, an inhibitor, or a cytotoxic factor to cells is easily read. This procedure has very few steps, has minimal manipulation of cells, and allows for good reproducibility. Alternatively, uptake of 3H-thymidine is used specifically to assay DNA synthesis, or as a more sensitive assay of cell proliferation for slow growing cells.

    Cell death occurs by lysis, necrosis, or apoptosis. Lysis is the destruction of the cell surface membrane such as by the action of an antibody and complement that makes holes in the membrane. Necrosis occurs through the action of toxic factors that act within the cell, such as irreversible inhibitors of protein, RNA or DNA synthesis, or mitotic poisons. Apoptosis is a programed cell death used by the body to remove damaged or unwanted cells and occurs during cytotoxic T cell killing and with some cancer chemotherapies. Apoptosis is characterized by early events such as expression of phosphotidylserine on the cell surface and fragmentation of the DNA, followed by loss of membrane integrity and mitochondrial function.

    The quantitative percentage of dying cells is determined microscopically or by flow cytometry using vital stains or DNA-binding dyes.

    High throughput measurement of cell death is performed by release of a label from cells prelabeled with a radiotracer, typically 51Cr, or a fluorescent or color marker. Alternatively, the fluorescent or colorimetric dye method described above is used.

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