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Assay Guidance Manual . Cytotoxicity Assays: In Vitro Methods to Measure Dead Cells. 2019

Riss, T., Niles, A., Moravec, R., Karassina, N. and Vidugiriene, J.

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)

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Pharm. Res. 128, 231-243. Globular adiponectin protects hepatocytes from tunicamycin-induced cell death via modulation of the inflammasome and heme oxygenase-1 induction. 2018

Khakurel, A., and Park, P.H.

Notes: The protective effect of globular adiponectin (gAcrp) in endoplasmic reticulum (ER) stress and cell death in hepatocytes was investigated. Treatment with gAcrp decreased caspase activation and lowered lactate dehydrogenase release as measured by the Caspase-Glo® 1 and CytoTox® 96 Non-Radioactive Cytotoxicity assays. These results show that gAcrp protects the liver from inflammation. (5253)

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Cell Death Dis. 9(2), 41. Sensitizing tumor cells to conventional drugs: HSP70 chaperone inhibitors, their selection and application in cancer models. 2018

Lazarev V.F., Sverchinsky D.V., Mikhaylova E.R., Semenyuk P.I., Komarova E.Y., Niskanen S.A., Nikotina A.D., Burakov A.V., Kartsev V.G., Guzhova I.V., Margulis B.A.

Notes: Heat shock protein 70 (Hsp70) inhibitors have shown substantial anticancer activity, however many are highly cytotoxic towards non-cancerous cells. This paper developed new drug discovery assays for compounds that reduce the chaperone activity of Hsp70. Chaperone and refolding activity were monitored using the Bright-Glo™ Luciferase Assay System. For the refolding assay, cells were heat shocked leading to luciferase denaturation. Active Hsp70 chaperone activity lead to refolding and luciferase signal. A novel compound, AEAC, showed two-fold lower cytotoxicity, as measured by the CytoTox 96® Non-Radioactive Cytotoxicity Assay, and inhibition of Hsp70 activity. (5173)

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Nat. Commun. 9(1), 1591. Multi-faceted immunomodulatory and tissue-tropic clinical bacterial isolate potentiates prostate cancer immunotherapy. 2018

Anker, J.F., Naseem, A.F., Mok, H., Schaeffer, A.J., Abdulkadir, S.A., and Thumbikat, P.

Notes: These authors investigated whether localized bacterial infection could be used to help stimulate an immune response and render prostate tumors susceptible to immune checkpoint inhibitors. They showed that an E.coli strain (CP-1) isolated from a prostate infection colonized prostate tumors and enhanced immune checkpoint inhibition. The Caspase-Glo® 3/7 Assay, CellTiter® AQueous ONE Solution and the CytoTox® 96 Assay were used to assess apoptosis, cell viability and cytotoxicity, respectively.  (5047)

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Mediators Inflammation , Article ID: 737310. Role of calprotectin as a modulator of the IL27-mediated proinflammatory effect on endothelial cells. 2015

Dorosz, S.A., Ginolhac, A., Kähne, T., Naumann, M., Sauter, T., Salsmann, A., and Bueb, J.-L.

Notes: Human Umbilical Vein Endothelial cells (HUVECs) were treated with IL-27, calprotectin, TNFα or combinations. Cell number per well was controlled by lysing the cells and measuring the LDH release with the CytoTox 96® Assay. Changes in gene expression were monitored by probe-based qPCR with cDNA made with the GoScript™ Reverse Transcription System using total RNA isolated using the ReliaPrep™ RNA Cell Miniprep System. Changes in protein levels upon treatment were evaluated by mass spec using Trypsin Gold for peptide analysis. (4598)

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PLos ONE 9, e98877. Induction of cell-mediated immune responses in mice by DNA vaccines that express hepatitis C virus NS3 mutants lacking serine protease and NTPase/RNA helicase activities. 2014

Ratnoglik, S.L., Jiang, D.-P., Aoki, C., Sudarmono, P., Shoji, I., Deng, L. and Hotta, H.

Notes: Primary splenocytes were isolated from mice immunized with an NS3 expression vector and examined for IFN-γ expression by RT-qPCR. Total RNA was isolated with the ReliaPrep™ RNA Cell Miniprep System and converted to cDNA with the GoScript™ Reverse Transcription System. The affect of wildtype and mutant NS3 on interferon-β promoter activity with an IFN-β promoter firefly luciferase vector and pRL-TK vector control was measured with the Dual-Luciferase® Reporter Assay System. LDH release was measured with the CytoTox 96® Cytotoxicity Assay in a cytotoxic T-lymphocyte assay using cultured splenocytes from the immunized mice. (4609)

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J. Biomol. Scr. 13, 591-608. High-throughput screening-based identification of paramyxovirus inhibitors. 2008

Yoon, J.-J., Chawla, D., Pall, T., Ndungu, M., Du, Y., Kurtkaya, S., Sun, A., Snyder, J.P. and Plemper, R.K.

Notes: The authors describe an HTS assay to screen for inhibitors of measles virus infection. The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess toxicity of all confirmed hit compounds from the primary screen. A luminescent assay was used to assess cell-to-cell fusion in the presence candidate compounds that appeared to inhibit entry. Effector cells that expressed the T7 polymerase and measles virus H and F envelope proteins were overlaid on target cells expressing firefly luciferase under the control of a T7 polymerase promoter. Inhibition of fusion should reduce luciferase expression compared to a positive fusion control. Ten of the eleven compounds tested caused a dose-dependent reduction in luciferase expression, suggesting they block viral entry into cells. Luminescence was detected using the Bright-Glo™ Assay System. (3933)

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J. Immunol. 179, 4829–4839. Aging up-regulates expression of inflammatory mediators in mouse adipose tissue. 2007

Wu, D., Ren, Z., Pae, M., Guo, W., Cui, X., Merrill, A.H. and Meydani, S.N.

Notes: The authors examined the role of ceramide and NF-κB in age-related adipose tissue inflammation in mice. Levels of inflammation-associated molecules, IL-1β, IL-6, TNF-α, COX-2 and peroxisome proliferator-activated receptor (PPAR)-γ mRNA, were quantified by quantitive PCR (qPCR). RNA was isolated from adipose tissues, and first-strand cDNA was synthesized using the Reverse Transcription System prior to qPCR. mRNA levels were also quantified in peritoneal macrophages grown in the presence of young adipocyte-conditioned medium and old adipocyte-conditioned medium. Viability of the primary adipocytes used in these experiments was confirmed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay and CytoTox 96® Non-Radioactive Cytotoxicity Assay. (3777)

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Am. J. Respir. Cell Mol. Biol. 34, 119–27. (R)-Albuterol elicits anti-inflammatory effects in human airway epithelial cells via iNOS. 2006

Chorley, B.N., Li, Y., Fang, S., Park, J-A. and Adler, K.B.

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)

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Biochemistry 43, 13348-13356. Lipidic carriers of siRNA: Differences in the formulation, cellular uptake, and delivery with plasmid DNA. 2004

Spagnou, S., Miller, A.D. and Keller, M.

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to determine the cytotoxic effect of lipophilic transfection reagents commonly used for siRNA transfection. Data are presented as the percent cell death observed in HeLa and IGROV-1 cells at 24 hours post-transfection. The researchers tested the effect of each reagent with and without siRNA during transfections.  (3178)

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Microbiology 150(Pt 4), 1079-84. The role of the Shigella flexneri yihE gene in LPS synthesis and virulence 2004

Edwards-Jones, B., Langford, P.R., Kroll, J.S., Yu, J.

Notes: Shigella flexneri serotype 5a virulent wild type or mutant strain was used to infect HeLa cultures. After 90 minutes, the level of LDH in the cell culture medium was assayed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay. (3166)

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Am. J. Pathol. 163(3), 869–878. Decorin inhibition of PDGF-stimulated vascular smooth muscle cell function: potential mechanism for inhibition of intimal hyperplasia after balloon angioplasty. 2003

Nili, N., Cheema, A.N., Giordano, F.J., Barolet, A.W., Babaei, S., Hickey, R., Eskandarian, M.R., Smeets, M., Butany, J., Pasterkamp, G., Strauss, BH.

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess cell necrosis after treated SMCs with decorin to induce platelet-derived growth factor receptor. (2986)

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Blood 102(4), 1435-1442. Ex vivo induction of multiple myeloma-specific cytotoxic T lymphocytes. 2003

Hayashi, T., Hideshima, T., Akiyama, M., Raje, N., Richardson, P., Chauhan, D., Anderson, K.C.

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess the cytotoxic T-lymphocyte activity of patients suffering multiple-myeloma against autologous primary MM cells. (2971)

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J. Neurochem. 85(6), 1412-20. Interleukin-18 induces expression and release of cytokines from murine glial cells: interactions with interleukin-1 beta. 2003

Wheeler, R.D., Brough, D., Le Feuvre, R.A., Takeda, K., Iwakura, Y., Luheshi, G.N., Rothwell, N.J.

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to measure cell death in mixed glia and microglia isolated from IL-18 or IL-1ß knockout mice. The cells were treated vehicle, IL-18, IL-1β or LPS for twenty-four hours before measurements were taken. (3139)

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Clin. Can. Res. 9, 1906-1916. Novel kidney cancer immunotherapy based on the granulocyte-macrophage colony-stimulating factor and carbonic anhydrase IX fusion gene 2003

Hernández, J.M., Bui, M.H.T., Han, K-r., Mukouyama, H., Freitas, D.G., Nguyen, D., Caliliw, R., Shintaku, P.I., Paik, S.H., Tso, C-L., Figlin, R.A., Belldegrun, A.S.

Notes: pGEM®-T Easy Vector was used to clone PCR products.  The CytoTox 96® Non-Radioactive Cytotoxicity Assay  was used to determine specific cytotoxicity of human dendritic cells that were transduced with recombinant adenoviruses containing the gene encoding a fusion protein of granulocyte-macrophage colony stimulating factor and carbonic anhydrase IX. (2674)

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J. Immunol. 170(12), 5919–5926. Potentiation of a tumor cell susceptibility to autologous CTL killing by restoration of wild-type p53 function. 2003

Thiery, J., Dorothee, G., Haddada, H., Echchakir, H., Richon, C., Stancou, R., Vergnon, I., Benard, J., Mami-Chouaib, F., Chouaib, S.

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess cell-mediated cytotoxicity when a tumor cell line treated with Fas-neutralizing antibody was exposed to tumor-infiltrating lymphocytes. (2917)

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Br. J. Haematol. 121(4), 592–596. Recombinant humanized anti-CD40 monoclonal antibody triggers autologous antibody-dependent cell-mediated cytotoxicity against multiple myeloma cells. 2003

Hayashi, T., Treon, S.P., Hideshima, T., Tai, Y.T., Akiyama, M., Richardson, P., Chauhan, D., Grewal, I.S., Anderson, KC.

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess antibody-dependent cell-mediated cytotoxicity using rhuCD40 mAb on B cells cocultured with effector mononuclear cells at various effector to target ratios. (2966)

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J. Mol. Med. 80(4), 233–242. Antiviral activity of artesunate towards wild-type, recombinant, and ganciclovir-resistant human cytomegaloviruses. 2002

Efferth, T., Marschall, M., Wang, X., Huong, S.M., Hauber, I., Olbrich, A., Kronschnabl, M., Stamminger, T., Huang, ES.

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess antiviral compound efficacy on cells infected with a GFP-expressing HCMV. Instead of assaying the media, the researches evaluated the residual cell layer. (2909)

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Nat. Med. 8, 358-365. CD39 is the dominant Langerhans cell-associated ecto-NTPDase: Modulatory roles in inflammation and immune responsiveness 2002

Mizumoto, N., Kumamoto, T., Robson, S.C., Sevigny, J., Matsue, H., Enjyoji, K., Takashima, A.

Notes: Pam-212 keratinocytes (murine) or freshly isolated epidermal cells were treated in PBS with a variety of skin irritants in an attempt to stimulate release of ATP from the cells. Supernatants from the cell cultures were examined for ATP concentrations using a  luciferin-luciferase assay and  for LDH release using Promega's CytoTox 96® Assay.  The percent -specific release of LDH was calculated  based on total LDH concentration detected in the supernatants after permeabilization with 0.3% Triton X-100. The authors conclude that injured keratinocytes release ATP. (2431)

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J. Gen. Virol. 83(Pt 5), 1013–1023. Direct targeting of human cytomegalovirus protein kinase pUL97 by kinase inhibitors is a novel principle for antiviral therapy. 2002

Marschall, M., Stein-Gerlach, M., Freitag, M., Kupfer, R., van, den, Bogaard, M., Stamminger, T.

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess inhibitors of a HCMV protein kinase on viral cytotoxicity. (2910)

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Emerging Infect. Dis. 8(8), 796-801. DNA vaccine expressing conserved influenza virus proteins protective against H5N1 challenge infection in mice. 2002

Epstein, S.L., Tumpey, T.M., Misplon, J.A., Lo, C.Y., Cooper, L.A., Subbarao, K., Renshaw, M., Sambhara, S., Katz, J.M.

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess the CTL activity of mouse splenocytes removed from mice immunized with a DNA vaccine and challenged with various H5N1 influenza virus strains. (2958)

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Nat. Neurosci. 5, 405-414. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways 2002

Hardingham, G.E., Fukunaga, Y., Bading, H.

Notes: Hipppocampal neurons were cultured for 10-12 days and then hypoxic or ischemic conditions were induced. Eight hours after this event acute cell death was quantified by measurements of LDH release using Promega's CytoTox 96® Non-Radioactive Cytotoxicity Assay. (2439)

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Mol. Cell. Neurosci. 19, 417-29. Gas1 is induced during and participates in excitotoxic neuronal death 2002

Mellström, B., Ceña, V., Lama, M., Perales, C., Gonzalez, C., Naranjo, J.R.

Notes: Transiently transfected primary neuronal cells expressing beta-galactosidase were treated with NMDA and stained with X-Gal using the Promega method. Cytotoxicity of the treated cells was analyzed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay. (2580)

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Am. J. Respir. Cell Mol. Biol. 26(4), 447–452. Neutrophil elastase induces MUC5AC gene expression in airway epithelium via a pathway involving reactive oxygen species. 2002

Fischer, B.M., Voynow, JA.

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess cellular cytotoxicity after respiratory cells were treated with neutrophil elastase and antioxidants. (2864)

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J. Immunol. 169(11), 6522–6529. Production of type I IFN sensitizes macrophages to cell death induced by Listeria monocytogenes. 2002

Stockinger, S., Materna, T., Stoiber, D., Bayr, L., Steinborn, R., Kolbe, T., Unger, H., Chakraborty, T., Levy, D.E., Muller, M., Decker, T.

Notes: The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to assess cell death when macrophage cells were infected with Listeria monocytogenes. (2871)

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