Cell-death assays

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. 

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. 

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. 

Apoptosis or programmed cell death is one of the regulatory mechanisms for the removal of unwanted cells. Apoptosis is induced by the stimulation of several different cell surface receptors in association with caspase activation. Apoptosis of a cell is thus a complicated process and can be assayed by various methods. Among widely used methods, the TUNEL assay is described here. 

The principle of the human skin model test is that the test material is apphed topically for up to 4h to a three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum comeum (outermost layer of the skin). The human skin models can come from various sources, but they must meet certain criteria. Corrosive materials are identified by their abdity to produce a decrease in cell viabdity (as determined, e.g., by using a dye reduction assay) below defined threshold levels at specified exposure periods. The principle of the test is in accordance with the hypothesis that corrosive chemicals are able to penetrate the stratum comeum (by diffusion or erosion) and are sufficiently cytotoxic to cause cell death in the underlying cell layers. 

The synthetic lethal effect between PARP inhibition and BRCAl/2 deficiency was first described in 2005 by Farmer et al and Bryant et al in preclinical studies (17, 18). BRCAl/2 deficient cell lines are more sensitive to PARP inhibition than BRCAl/2 proficient cell lines in colony formation assays and in xenograft models (17, 18). BER and HR are DNA repair pathways that carry out the repair of SSB and DSB, respectively. Inhibition of either pathway alone is not lethal, but loss of both pathways causes cell death (Fig. 2). Mechanistically, PARP inhibition blocks the activity of BER and leads to DNA SSB accumulation inside cells. When cells enter the next round of DNA replication, these DNA SSBs are converted to DNA DSBs which can be efficiently repaired through... 

In this study, annexin V/PI assay was used to detect cell death by flow cytometry (30) (see Note 11). 

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