A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers. Anal. Biochem. 366 (2): 197206. doi: 10.1016j.ab.2007.04.007. PMID 17512890.Examples of toxic agents are an immune cell or some types of venom, e.g.
The cells can stop actively growing and dividing (a decrease in cell viability), or the cells can activate a genetic program of controlled cell death ( apoptosis ). Cells that undergo rapid necrosis in vitro do not have sufficient time or energy to activate apoptotic machinery and will not express apoptotic markers. Apoptosis is characterized by well defined cytological and molecular events including a change in the refractive index of the cell, cytoplasmic shrinkage, nuclear condensation and cleavage of DNA into regularly sized fragments. Cells in culture that are undergoing apoptosis eventually undergo secondary necrosis. They will shut down metabolism, lose membrane integrity and lyse. Researchers can either look for cytotoxic compounds, if they are interested in developing a therapeutic that targets rapidly dividing cancer cells, for instance; or they can screen hits from initial high-throughput drug screens for unwanted cytotoxic effects before investing in their development as a pharmaceutical. Compounds that have cytotoxic effects often compromise cell membrane integrity. Vital dyes, such as trypan blue or propidium iodide are normally excluded from the inside of healthy cells; however, if the cell membrane has been compromised, they freely cross the membrane and stain intracellular components. One molecule, lactate dehydrogenase (LDH), is commonly measured using LDH assay. LDH reduces NAD to NADH which elicits a colour change by interaction with a specific probe. Protease biomarkers have been identified that allow researchers to measure relative numbers of live and dead cells within the same cell population. The live-cell protease is only active in cells that have a healthy cell membrane, and loses activity once the cell is compromised and the protease is exposed to the external environment. The dead-cell protease cannot cross the cell membrane, and can only be measured in culture media after cells have lost their membrane integrity. This assay measures the reducing potential of the cell using a colorimetric reaction. Viable cells will reduce the MTS reagent to a colored formazan product. A similar redox-based assay has also been developed using the fluorescent dye, resazurin. In addition to using dyes to indicate the redox potential of cells in order to monitor their viability, researchers have developed assays that use ATP content as a marker of viability. Such ATP-based assays include bioluminescent assays in which ATP is the limiting reagent for the luciferase reaction. A possible combination is LDH-XTT-NR (Neutral red assay)-SRB which is also available in a kit format. This technology is referred to as electric cell-substrate impedance sensing (ECIS). Label-free real-time techniques provide the kinetics of the cytotoxic response rather than just a snapshot like many colorimetric endpoint assays. Lymphocyte-mediated cytotoxicity, on the other hand, does not have to be mediated by antibodies; nor does complement-dependent cytotoxicity (CDC), which is mediated by the complement system. Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. Assay Drug Dev Technol. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J. Immunol. Methods. A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers.
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