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: Antisense fluorophore octaarginine peptide nucleic acid (PNA) conjugates are investigated cellular activity using a photochemical internalization (PCI) drug delivery method. PCI utilizes light to generate reactive oxygen species that can damage the endosomal membrane and lead to drug release. Specifically, cellular localization and antisense activity was monitored for two conjugates, tetramethylrhodamine and Alexa Fluor 555. Luminescence and cell viability were measured using the Bright-Glo™ Assay and CellTiter-Glo® Assay, respectively. (5172)

Notes: The effects of hydrogen sulfide on inflammasome activation were investigated. Lactate dehydrogenase levels and caspase activation were monitored using the CytoTox-ONE™ and Caspase-Glo® assays. Hydrogen sulfide was shown to decrease caspase activation and inflammasome activity both in vitro and in vivo. (5147)

Notes: Cell viability was used as a reference parameter for acute cytotoxicity of airborne particulate matter and was assessed using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. Luminescent signal was measured using the GloMax^® Discover System. (5085)

Notes: Human primary hepatocytes at a density of 2 × 10⁴ were seeded into 96-well collagen-coated plates, incubated overnight and then stimulated with two compounds. After 48 hours, the levels of NAD+ was assessed using the NAD/NADH-Glo™ Assay. HepG2 and AML12 cells (5 × 10³) were treated with test compounds for 4 hours in a 384-well plate. Cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay and cell necrosis evaluated using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. (4953)

Notes: The authors demonstrate that the X-linked inhibitor of apoptosis (XIAP) drives anti-EGFR and anti-HER antibody resistance in inflammatory breast cancer both through its effect on caspase and suppression of ROS accumulation. They use the CytoTox ONE Homogeneous Membrane Integrity Assay to assess cytotoxicity in wild type SUM149 and SUM190 cells, and  rSUM149 and rSUM190 cells (cell lines that have acquired resistance to treatment agents) upon treatment with cytotoxic agents. The Caspase-Glo® 3/7 Assay was used to assess apoptosis in wild type cells, rSUM149 and in cells over expressing XIAP (treated and untreated). (4726)

Notes: The authors sought to determine the function of ceramide and ceramide synthase (CerS6) in mitochondria during post-natal brain development. They derived oligodendrocytes (OLs) from cultured, dissociated rat neonatal cortices. To determine if CerS6 plays in cell death, OLs were treated with glutamate to induce excitotoxicity, and cell death was assessed using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. To determine if the glutamate-induced excitotoxicity is dependent on ceramide or CerS6, OLs were treated with glumate in the presence or absence of the CerS6 inhibitor, fumonisin B1. To assess the mechanism of cell death resulting from ceramide-dependent glutamate toxicity in OLs, caspase activation during glutamate treatment was measured using the Apo-ONE^® Homogeneous Caspase-3/7 Assay. (4171)

Notes: These authors characterized inflammatory caspase substrates using an enzymatic enrichment method for caspase 1,-4 and -5 cleaved proteins and mass-spectrometry. To confirm that caspase-1 cleaved the putative substrates, some substrates were expressed and fluorescently labeled using the TNT® T7 Transcription/Translation System and the FluorTect™ Green_Lys System. The proteins were then treated with recombinant caspase-1, and the progression of the reaction tracked via SDS-PAGE. The authors also used the CytoTox™ ONE Homogeneous Membrane Integrity Assay to track membrane permeabilization in relation to caspase cleavage and IL-1B release. (4252)

Notes: The authors describe a multiplexed in vitro assay to detect nephrotoxicity and gain information about mechanism of cell death in HK-2 (human kidney-2) cells. The multiplexed assay involved an LDH assay to detect necrosis, a caspase-3/7 assay to detect apoptosis, a reazurin assay to assess metabolic state, and a DNA dye staining assay to monitor nuclear morphology. (4002)

Notes: The authors of this study sought to identify inhibitors of myelin-reactive T-cell proliferation. They used spleen cells isolated from transgenic mice expressing T-cell receptors that specifically recognize myelin proteolipid protein residues 139-151 (PLP_139-151). Spleen cell suspensions were prepared from the transgenic mice and exposed to PLP in the presence of test compounds. Proliferation was assessed relative to positive and negative controls using the CellTiter-Glo® Luminescent Cell Viability Assay. Of the 41,184 compounds screened, 302 hits were obtained. These 302 compounds were next evaluated for nonspecific cytotoxicity using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay, and compounds causing nonspecific toxicity were eliminated from further consideration. Z´-factor values for the primary screen were robust (> 0.5). (3733)

Notes: NHBE cells were seeded and grown in 12-well plates in growth medium until 60–79% confluency. Medium was replaced with medium free of antibiotics or serum to induce quiescence. Transfection reagent and DNA were prepared as follows: 2µl of FuGENE® 6 reagent was added to 48µl of serum- and antibiotic-free medium and incubated at room temperature for 5 minutes. Next 0.1nmol of 21-bp iNOS siRNA was added and incubated for 15 minutes. 50µl of the transfection complex was added to each well for a final concentration of 0.45% FuGENE® 6 reagent and 225nM iNOS siRNA. Cells were incubated at 37°C for 24 hours.

To determine what, if any, protein kinases were involved in (R)-albuterol upregulation of iNOS message, inhibitors of several protein kinases were used to treat the NHBE cells. Because, protein kinase C inhibitors appeared to attenuated the (R)-albuterol-mediated iNOS expression, the PepTag® Non-Radioactive PKC Assay was used to measure protein kinase C activity in response to (R)-albuterol treatment.

All reagents were tested for cytotoxicity to NHBE cells using an LDH release assay. (4273)

Notes: A key component in the pathogenesis of Alzheimer's disease is cerebral accumulation of amyloid-beta protein (Aβ). Aβ is produced by proteolysis of amyloid-β-protein precursor (APP) by ß- and gamma-secretases, thus these enzymes are considered important drug targets for Alzheimer's disease. Existing assays for assessing inhibition of alpha-, beta- and gamma-secretases include HPLC or ELISA assays that are cumbersome, expensive and not well-suited to high-throughput screening. The authors developed a luciferase reporter system to identify new molecules that inhibit APP processing. They then successfully interfaced this sensitive, specific and quantitative assay with a high-throughput screen, useful for identifying both inhibitors and stimulators of APP processing. (3775)

Notes: In this study, the effect of arginosuccinate synthase (AS) on nitric oxide (NO) signaling was explored in bovine aortic endothelial cells. Expression of arginosuccinate synthase was knocked down using in-vitro-transcribed siRNAs. Using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay and the Apo-ONE® Homogeneous Caspase-3/7 Assay, it was found that reduction of AS leads to the induction of apoptosis. Detection was performed on a BMG FLUOstar spectrofluorometer. Supplying the cells with an NO donor decreased apoptosis induction. The regulatory role NO has on apoptosis is not known. (3065)

Notes: H69AR cells were seeded 10⁴ into wells of a 96-well microtiter plate. After a 24-hour incubation, the medium was replaced with new media with doxorubicin (DOX), liposomes containing DOX, and/or sense or antisense oligonucleotides. After a 48 hour incubation, the plates were assayed using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. (3161)

Notes: Cells were exposed to medium of 300, 540, and 620 mosmol/kg of H₂O for 0.5, 2, 6, 12 and 24 hours. Cell integrity was assayed using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. (3160)

Notes: After seeding 1 x 10⁵ cells/well in 96-wellplates, HeLa cells were allowed to attach for 24 hours. Cells were then treated with either media or WY-14643 (5-100 µM) in media for an hour before stimulation with 1 mM H₂O₂ (final concentration). Twelve hours later, hepatocyte injury was analyzed using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. (3167)

Notes: Mf4/4 cells seeded at 10⁶ cells/well of six-well plates in medium without antibiotics. After 15 hours, the cells were infected with multiplicity of infection of 50 with the Yersinia enterocolitica YopPE- or YopPE- strains that were complemented with wild type (YopEWT) or mutant YopE (YopEM). The cells were analyzed for cytotoxicity after 24 hours using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. (3158)

Notes: The effects of tamoxifen and vinblastine were assayed on HepG2 and HL-60 cells using a variety of Promega’s cell-based assays. The CellTiter-Glo^® Luminescent Cell Viability Assay, CellTiter 96^® AQueous One Solution Cell Proliferation Assay, and CellTiter-Blue^® Cell Viability Assay were used to test the effects of 0-100µM tamoxifen on HepG2 cell viability after an incubation period ranging from 0-24 hours. Cell membrane integrity was assessed with the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. The Caspase-Glo^® 3/7 Assay was also used to measure Caspase 3/7 activity after 0-100µM tamoxifen treatment. (3189)

Notes: The effects of bromodichloromethane (BDCM), a byproduct of water disinfection processes, on trophoblast cells isolated from term human placentas were investigated. The CytoTox-ONE™ Homogeneous Membrane Integrity Assay was used to assess cell membrane integrity and LDH release after trophoblasts were exposed to BDCM for 24 hours. Data was expressed in arbitrary fluorescence units. Ultimately, little difference was observed between BDCM-treated and untreated trophoblasts. (2756)

Notes: The CytoTox-ONE™ Homogeneous Membrane Integrity Assay was used to assess the cytotoxic effects of tranexamic acid (t-AMCHA) on neural human normal progenitor (NHNP) or normal human dermal fibroblasts (NHDF) cells. Varying concentrations of t-AMCHA (0.0 mM, 31.25 mM, 150 mM, 300 mM, and 450 mM) were added to 70-80% confluent cells, which were then cultured for 8 hours. t-AMCHA showed no toxic effect on either cell line.  (3031)

Notes: Mouse hepatocytes (AML-12 cell line) were assayed for cytotoxicity to 1 mmol/L hydrogen peroxide with and without 20ng/mL IL-13 using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. For these assays 1 x 10⁵ cells/well were seeded in 96-well microplates and cultured for 24 hours before IL-13 treatment. One hour later, hydrogen peroxide was added to the cultures. The cells were then cultured for an additional 24 hours before analysis with the CytoTox-ONE™ Homogeneous Membrane Integrity Assay.  (3030)

Notes: Primary hepatocytes from Sprauge-Dawley rats were incubated with various concentrations of tert-butylhydroperoxide to induce oxidative stress and then cell viability, cytotoxicity and apoptosis were assayed with Promega's CellTiter 96 AQueous One Solution Cell Proliferation Assay, CellTiter-Blue Cell Viability Assay, CellTiter-Glo Luminescent Cell Viability Assay, CytoTox-ONE Homogeneous Membrane Integrity Assay, Apo-ONE Homogeneous Caspase-3/7 Assay and assay systems. (2969)