Cell death, measurement

One type of test measures the inhibition of respiration of bacterial cells on pieces of pig skin by the substance under test. Here, the factor of correlation between cell death and cessation of respiration should be borne in mind. 

It is well accepted that MDMA produces 5-HT depletions in rat CNS, but much less attention has been devoted to the effects of MDMA on established markers of neurotoxicity such as cell death, silver-positive staining, and reactive gliosis. Support for the hypothesis of MDMA-induced axotomy relies heavily on immunohistochemical analysis of 5-HT levels, which could produce misleading results if not validated by other methods. For example, MDMA-induced loss of 5-HT could be due to persistent adaptive changes in gene expression or protein function, reflecting a state of metabolic quiescence rather than neurotoxic damage. Table 7.3 summarizes the effects of MDMA on hallmark measures of neurotoxicity. 

Before further testing and to confirm that the compounds are cytotoxic rather than merely interfering with the Alamar blue indicator dye, they are re-bioassayed using two other indicator dyes. Calcein-AM is a fluorescent dye that measures changes in cell mem brane permeability, an indicator for one of the penultimate steps of cell death, uci e e measures the amount of adenosine triphosphate (ATP) synthesis in a chemilurmne assay. For some compounds, cell death was also confirmed by microscopic exami Papanicolaou-stained cell preparations.11... 

This procedure, which complements other methods for distinguishing apoptotic and necrotic cell death, employs annexin V-PE as a marker for early apoptotic cells, and 7-AAD for late apoptotic or necrotic cells. Although other versions of this assay have used annexin V-fluorescein together with PI, that combination precludes the use of a third fluorescence color to measure an additional parameter, such as a phenotypic marker, because PI, unlike 7-AAD, has a broad emission spectrum that includes both orange and red fluorescence. 

Sublethal Measurement as opposed to measurement of cell death is required. Measurement of the noxious substance or a signal transduction event may be too early in the pathogenesis if cell injury as opposed to measurement of adaptive and adverse effects... 

Jester, J.V, Ling, J., HarbeU, J. Measuring depth of injury (DOI) in an isolated rabbit eye irritation test (IRE) using biomarkers of cell death and viability. Toxicol In Vitro 24(2), 597-604 (2010). Epub 2009 Oct 24... 

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