Notes: This Assay Guidance Manual provides a resource to scientists optimizing assays used in drug discovery and development. The cytotoxicity chapter outlines several assays to detect dead cells, including LDH-Glo™, CytoTox-Glo™ and CellTox™ Green Cytotoxicity Assays, CellTiter-Glo® Luminescent Cell Viability Assay, CytoTox 96® Non-Radioactive Cytotoxicity Assay and CytoTox-ONE™ Homogeneous Membrane Integrity Assay. (5198)

Notes: The CytoTox-Glo™ Cytotoxicity Assay was used as the readout for a cytotoxic T lymphocyte assay involving effector and target cells. (5042)

Notes: The post-transcriptional modification of neddylation helps regulate protein activity, stability and localization. NanoLuc® Binary Technology (NanoBiT) was used to monitor covalent neddylation of Cul1 in a cellular context. SmBiT-Nedd8 and Cul1-LgBiT were expressed in HEK293 cells and luminescence was monitored as a response to a Nedd8-Cul1 covalent protein modification. Further, neddylation was monitored in the presence of both inhibitors and activators, and a concentration- and time-dependent response in luminescence was observed. (5076)

Notes: Researchers evaluated the treatment of hybridomas with macrophage-conditioned medium using the CytoTox-Glo™ Assay to monitor cell cytotoxicity and the Caspase-Glo^® 3/7 Assay to monitor caspase activation. (5032)

Notes: Researchers studied the relationship between Reactive Oxygen Species, cell movement and cell death using ViaFect™ Transfection Reagent for transient transfections and CytoTox-Glo™ Cytotoxicity Assay to assess cell death. (5041)

Notes: Researchers evaluated the impact of inhibiting autophagy on cells in vitro using cell viability, cytotoxicity and caspase activity assays. (5033)

Notes: The ApoTox-Glo™ Triplex and CytoTox-Glo™ Cytotoxicity Assays were used to evaluate cell viability, cytotoxicity and apoptosis following cell treatments. (5040)

Notes: These authors investigated the ability of Phyllanthus plant extracts to affect the metastatic activity of human lung (A549) and breast (MCF-7) cancer cell lines. They initially used CellTiter 96® AQ_ueous Non-Radioactive Cell Proliferation Assay and the GloMax®-Multi Detection System absorbance module to determine cytotoxicity of various Phyllanthus extracts. After determining the effective dose, the authors investigated the ability of these plant compounds to inhibit/reduce metastatic activity. They then evaluated the mechanism of cell death in treated cells using the Caspase-Glo® 3/7 Assay and the Glomax® Multi Detection System luminescence module to measure caspase activity, and the CytoTox-ONE™ Homogeneous Membrane Integrity Assay to measure LDH activity. (4196)

Notes: These authors investigated the relationship between VEGF and oxidative stress related to preeclampsia. They showed that VEGF activates Nrf2 in an ERK1/2-dependent manner, protecting against oxidative stress. They first used a dual-luciferase reporter assay and a pGL3-ARE vector construct to show that VEGF activates ARE in the cytotrophic cell line BeWo. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase® reporter assay system and the GloMax®-96 microplate luminometer. The authors then showed that inactivation of the transcription factor Nrf2 by shRNA abolished this VEGF-dependent ARE activation. To determine whether Nrf2 protected BeWo cells from oxidative stress, cells were pretreated with VEGF and then exposed to H₂O₂ before monitoring cell viability and cytotoxicity. Cytotoxicity assays were performed using the CytoTox-Glo™ Assay. (4199)

Notes: Dysregulation of the NF-kB pathway has been associated with the formation of a wide variety of tumors and other cancers, as well as diseases, including chronic inflammation and immunodeficiency. Because of the association of constitutive NF-kB regulation and tumors, inhibition of the NF-kB pathway by small molecule antagonists was thought to be a means of reversing or halting the growth and spread of tumors. The authors screened compounds from a database (the NCGC Pharmaceutical Collection or NPC) of small molecule compounds: 52% of the compounds have been approved for human or animal use by the FDA, 22% were drugs approved for use in Europe, and another 25% either drugs approved in other countries or compounds that have been tested in clinical trials. The database served as a source from which to rapidly and efficiently identify already approved drugs that inhibited NF-kB. They used a quantitative high-throughput screening format. To identify small molecules that could inhibit the NF-kB pathway, the compounds were initially screened using a cell-based NF-kB lactamase reporter gene assay, with TNFalpha and MG132 as positive controls. (TNFalpha induced NF-kB coupled beta-lactamase activity, while MG132 blocked TNFalpha induction NF-kB-coupled beta-lactamase activity.) After several rounds of screening, 20 compounds were further studied for their NF-kB inhibition, with NF-kB luc2P HEK293 cells. After a concordance rate of 95% between the luciferase and beta-lactamase tests, compounds were additionally examined for their ability to affect caspase 3/7 activity, for the ability to disrupt the electrochemical gradient across the mitochondrial membrane in relation to cellular apoptosis, as well as tests of the inhibitors on cancer cell viability and affects on LDH release, an indicator of cell necrosis.
(4049)

Notes: The authors of this paper describe an assay that uses protease biomarkers to assess cell viability and cell death simultaneously in a population of cells. The assay detects an ubiquitous protease activity that is associated with live cells and a second protease activity that is associated with cells that have lost membrane integrity. The readouts are either fluorescent or fluorescent and luminescent. The assay can be performed in multiplex with other assays, such as caspase assays, to gain additional information on the cell population, and it is amenable to high-throughput screening. (3927)