Death Assay

XIAPcontaining calls show resistance to apoptosis 500 compounds were tested in 

XIAP antagonists should promote cell death TRAiL  

31 compounds were shown to exhibit the abiJity to induce cell death at greater than 50% 

Laboratories, PolarStar Fluorometer. Using this assay, out of 500 compounds tested, 31 compounds were shown to exhibit the ability to introduce cell death greater than 50 %. 

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Prepare all media and handle cells in aseptic conditions except during the cell death assay. 

Many of the cell-death assays described above are readily performed by flow cytometry... 

Kepp, O., GaUuzzi, L., Lipinski, M., Yuan, J., and Kroemer, G. 2011. Cell death assays for drug discovery. Nat Rev Drug Discov, 10, 221-237. 

Apoptosis and necrosis were detected using the Cell Death Detection EL1SA kit, Roche (Version 11.0), a photometric enzyme-immunoassay for the qualitative and quantitative in vitro determination of cytoplasmic histone associated DNA fragments after induced cell death. Assay is based on a quantitative sandwich enzyme immunoassay principle using mouse monoclonal antibodies directed against DNA and histones that allows specific determination of mono- and oligonucleosomes in the cytoplasmatic fraction of the cell. 

Buenz, E. J. Limburg, P. J. Howe, C. L. A high-throughput 3-parameter flow cytometry-based cell death assay. Cytometry 2007, 71A, 170-173. 

Methods to Detect and Quantitate Viral Agents in Fluids. In order to assess the effectiveness of membrane filtration the abihty to quantitate the amount of vims present pre- and post-filtration is critical. There are a number of techniques used. The method of choice for filter challenge studies is the plaque assay which utilizes the formation of plaques, localized areas in the cell monolayer where cell death caused by viral infection in the cell has occurred on the cell monolayer. Each plaque represents the presence of a single infectious vims. Vims quantity in a sample can be determined by serial dilution until the number of plaques can be accurately counted. The effectiveness of viral removal may be determined, as in the case of bacterial removal, by comparing the vims concentration in the input suspension to the concentration of vims in the effluent. 

Potenza MN, Gold SJ, Roby-Shcmkowitz A, et al Effects of regulators of G protein-signaling proteins on the functional response of the mu-opioid receptor in a mel-anophore-based assay. J Pharmacol Exp Ther 291 482 91, 1999 Quaglio G, Talamini G, Lechi A, et al Study of 2708 heroin-related deaths in northeastern Italy 1985—98 to establish the main causes of death. Addiction 96 1127— 1137, 2001... 

The concept of biomarkers is illustrated in Figure 4.4. As the dose of a chemical increases, the organism moves from a state of homeostasis to a state of stress. With further increases in dose, the organism enters first the state of reversible disease, and eventually the state of irreversible disease, which will lead to death. In concept, all of these stages can be monitored by biomarker assays (lower part of conceptual diagram). 

Mouse Bioassay. The mouse is the traditional animal of choice for detecting biological activity due to STX and TTX. Mice receive an intraperitoneal injection of sample and are observed for symptoms of intoxication, i.e., dypsnea, convulsions, and death. This method is effective for detecting biological activity of STX and TTX in numerous samples. For the standard STX assay, one mouse unit is defined as that quantity of STX injected i.p. in 1 ml solution that will... 

Tissue Culture Assay. Kogure et al. (48) report a novel tissue culture assay for detecting several types of sodium channel blockers. The mouse neuroblastoma cell line ATCC CCL 131 is grown in RPMI 1640 supplemented with 13.5% fetal bovine serum and 100 pg/ml gentamycin, in an atmosphere of 5% C0 95% air at 37 C. Ninety-six well plates are seeded with 1 x 10 cells in 200 pi of medium containing 1 mM ouabain and 0.075 mM veratridine. Veratridine and ouabain cause neuroblastoma cells to round-up and die. In the presence of sodium channel blockers (e.g., TTXs or STXs), the lethal action of veratridine is obviated and cells retain normal morphology and viability. An important feature of this assay is that a positive test for sodium channel blockers results in normal cell viability. Since bacterial extracts can contain cytotoxic components, this assay offers an advantage over tests that use cell death as an endpoint. The minimum detectable level of TTX is approximately 3 nM, or approximately 1/1000 mouse unit. 

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