Journal Article•
A rapid, quantitative fluorescence assay for cell damage by cytotoxic antibodies.
01 May 1970-Journal of Immunology (American Association of Immunologists)-Vol. 104, Iss: 5, pp 1303-1306
TL;DR: The laboratory sought another approach to differential fluorescent staining of cells, which would still allow machine-reading of the assays, and chose to screen a number of fluorescent probes, compounds whose fluorescence is enhanced when bound to protein or nucleic acids.
Abstract: Our laboratory recently (1) described a fluorochromatic assay for cell damage by cytotoxic alloantibody, which was based on retention of fluorescein by intact cells after its hydrolysis from fluorescein diacetate esters (FDA) (2) Although the method gave reproducible results in our hands, it was slow and always subject to error, since traces of complement used in the reaction were capable of hydrolyzing FDA, thus yielding erroneously high values of cell viability This was not satisfactory and accordingly we sought another approach to differential fluorescent staining of cells, which would still allow machine-reading of the assays Rather than looking for other compounds that were enzymatically changed from non-fluorescent to fluorescent, we chose to screen a number of fluorescent probes (3), compounds whose fluorescence is enhanced when bound to protein or nucleic acids Such compounds were initially screened by observing fresh and heat-damaged lymphocytes, bathed in solutions of various probes, with the fluorescence microscope
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TL;DR: In this paper, a rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension.
Abstract: A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.
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TL;DR: A hemocytometer‐based trypan blue dye exclusion cell quantitation and viability assay was compared with a similar assay using simultaneous fluorometric staining with fluorescein diacetate and propidium iodide, and the fluorometric assay was considered a better choice for the evaluation of cell viability.
Abstract: A hemocytometer-based trypan blue dye exclusion cell quantitation and viability assay was compared with a similar assay using simultaneous fluorometric staining with fluorescein diacetate and propidium iodide. Viable and nonviable cell densities were measured, and culture viability was calculated both during the normal growth cycle of a murine hybridoma and in response to the application of millimolar concentrations of either tert-butyl hydroperoxide or ferrous iron. During the early phase of rapid hybridoma cell growth, assay-based differences in viable cell density were not significant. As the culture aged, the trypan blue dye exclusion assay significantly overestimated cell viability, thereby underestimating nonviable cell density and yielding an erroneous estimation of the overall viability of the culture. Because of its lack of ambiguity in the identification of stained, nonviable cells and its resulting increased accuracy in the estimation of culture viability, the fluorometric assay was considered a better choice for the evaluation of cell viability.
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