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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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Cell Chemical Biology 25, 1337–1349. APD-containing cyclolipodepsipeptides target mitochondrial function in hypoxic cancer cells. 2018

Jacobsen, K.M., Villadsen, N.L., Tørring, T., Nielsen, C.B., Salomón, T., Nielsen, M.M., Tsakos, M., Sibbersen, C., Scavenius, C., Nielsen, R., Christensen, E.I., Guerra, P.F., Bross, P., Pedersen, J.S., Enghild, J.J., Johannsen, M., Frøkiær, J., Overgaard, J., Horsman, M.R., Busk, M. and Poulsen, T.B.

Notes: Hypoxic cancer cells constitute a major challenge in chemo- and radiotherapy. The researchers present evaluation of APD-CLD compounds that induce selective cell death in hypoxic cancer cells by impeding mitochondrial function. The RealTime-Glo™ Annexin V Apoptosis Assay, CellTiter-Blue® Cell Viability Assay, CellTox™ Green Real-Time Cytotoxicity Assay and GSH-Glo™ Glutathione Assay were used to assess mechanism of action. (5168)

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Cancer Res. 78(13), 3432-3444. High USP6NL levels in breast cancer sustain chronic AKT phosphorylation and GLUT1 stability fueling aerobic glycolysis. 2018

Avanzato, D., Pupo, E., Ducano, N., Isella, C., Bertalot, G., Luise, C., Pece, S., Bruna, A., Rueda, O.M., Caldas, C., Di Fiore, P.P., Sapino, A., and Lanzetti, L.

Notes: The role of USP6NL in metabolic rewiring in breast cancer was investigated. The CellTox™ Green and CellTiter-Glo® assays were used to measure cell toxicity and ATP levels, respectively. Depletion of USP6NL in certain breast cancer cell lines lead to decreased glucose uptake as measured by the Glucose Uptake-Glo™ assay. USP6NL was shown to be involved in activation of glycolysis in breast cancer cells. (5150)

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Cancer Res. 78, 3432-3444. High USP6NL Levels in Breast Cancer Sustain Chronic AKT Phosphorylation and GLUT1 Stability Fueling Aerobic Glycolysis. 2018

Avanzato, D., Pupo, E., Ducano, N., Isella, C., Bertalot, G., Luise, C., Pece, S., Bruna, A., Rueda, O.M., Caldas, C., Di Fiore, P.P., Sapino, A. and Lanzetti, L.

Notes: Cell toxicity and ATP cell content were evaluated with the CellTox™ Green Cytotoxicity Assay and CellTiter-Glo® Luminescent Cell Viability Assay. Glucose uptake was measured with the Glucose Uptake-Glo™ Assay. (5004)

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Veterinary Microbiology 219, 226–33. The nucleoside analog GS-441524 strongly inhibits feline infectious peritonitis (FIP) virus in tissue culture and experimental cat infection studies. 2018

Murphy, B.G., Perron, M., Murakami, E., Bauer, K., Park, Y., Eckstrand, C., Liepnieks, M., and Pedersen, N.C.

Notes: Antiviral therapy is effective at treating feline infectious peritonitis virus, in both in vivo and in vitro experiments. Cytotoxicity of the antiviral therapy in CRFK cells was evaluated using CellTox Green™ Assay. (5036)

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J. Cell Biol. 216, 1567-77. Oncogenic β-catenin and PIK3CA instruct network states and cancer phenotypes in intestinal organoids.


Riemer, P., Rydenfelt, M., Marks, M., van Eunen, K., Thedieck, K., Herrmann, B.G., Blüthgen, N., Sers, C., and Morkel, M.


Notes: These authors used organoid cultures to study changes in cancer-related signaling networks. Metabolic activity in the 3D cultures was measured using the RealTime Glo™ MT Cell Viability Assay and the CellTox™ Green Dye.


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Nat. Commun. 8(1), 732. Control of leucine-dependent mTORC1 pathway through chemical intervention of leucyl-tRNA synthetase and RagD interaction. 2017

Kim, J.H., Lee, C., Lee, M., Wang, H., Kim, K., Park, S.J., Yoon, I., Jang, J., Zhao, H., Kim, H.K., Kwon, N.H., Jeong, S.J., Yoo, H.C., Kim, J.H., Yang J.S., Lee, M.Y., Lee, C.W., Yun, J., Oh, S.J., Kang, J.S., Martinis, S.A., Hwang, K.Y., Guo, M., Han, G., Han, J.M., and Kim, S.


Notes: These authors evaluated the effect of BC-LI-0186, a compound that inhibits leucine-dependent mTORC1 activity, on cell growth and death using human colon cancer SW620 cells. They showed that the compound inhibited growth of cancer cells expressing drug-resistant MTOR mutations. Cell viability and cytotoxicity were determined in SM620 cells stably expressing the red fluorescent protein CellPlayer™ NucLight Red (Essen BioScience) and treated with BC-LI-0186 diluted in media containing CellTox™ Green Dye (Promega). Viable (Red) and dead (green) signals were acquired every 2 h and quantification performed using an IncuCyte™ Zoom basic analyzer. (5043)

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Exp. Cell Res. 355(1), 47–56. Gas6-Axl signaling in presence of Sunitinib is enhanced, diversified and sustained in renal tumor cells, resulting in tumor-progressive advantages. 2017

Gustafsson, A., Fritz, H.K.M., and Dahlbäck, B.

Notes: These authors investigated Gas6-mediated Axl signaling in Clear Cell Renal Cell Carcinoma (CCRCC), a cancer that is associated with development of chemoresistance and recurrence. They report that Axl signaling was enhanced in the presence of Sunitinib, possibly contributing to acquired chemoresistance. As part of the study the CellTox™ Green and CellTiter-Glo® 2.0 Assays were used to assess late apoptosis and cell viability, respectively. (5037)

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PLos ONE 12, e0177902. Real-time cell toxicity profiling of Tox21 10K compounds reveals cytotoxicity dependent toxicity pathway linkage 2017

Hsieh, J. H., Huang, R., Lin, J. A., Sedykh, A., Zhao, J., Tice, R. R., and Auerbach, S. S.

Notes: Chemical-induced cytotoxicity was investigated in HEK293 and HepG2 cells using two real-time assay technologies: the Promega RealTime-Glo™ MT Cell Viability Assay and the Promega CellTox™ Green Cytotoxicity Assay.  (4870)

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Biochim. Biophys. Acta 1864(6), 947-956. Resveratrol-induced autophagy is dependent on IP3Rs and on cytosolic Ca2. 2017

Luyten, T., Welkenhuyzen, K., Roest, G., Kania, E., Wang, L., Bittremieux, M., Yule, D.I., Parys, J.B., and Bultynck, G..

Notes: These authors investigated whether autophagy induced by resveratrol requires IP3Rs and Ca2+ signaling. Resveratrol augmented autophagic flux in a time-dependent manner in HeLa cells, but induction of autophagy was completely abolished in the presence of the intracellular Ca2+-chelating agent, BAPTA-AM. As part of the study cytotoxicity of BAPTA-AM and resveratrol was measured in HeLa cells using the CellTox™ Green Assay. (5044)

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Biochem. J. 474(10), 1591-1602. The human cancer cell active toxin Cry41Aa from Bacillus thuringiensis acts like its insecticidal counterparts. 2017

Krishnan, V., Domanska, B., Elhigazi, A., Afolabi, F., West, M.J., and Crickmore, N.

Notes: This study investigated the effect of the Bacillus thuringiensi toxins parasporin 3 on HepG2 cells. Understanding how Bt toxins target human cell lines has implications for the risk assessment of products containing these toxins, such as transgenic, insect-resistant plants. The authors used the CellTiter®-Blue, CellTiter®-Glo, Caspase-Glo® and ROS-Glo™ assays to assess viability, apoptosis induction, and oxidative stress in cultured cells exposed to the toxin. (5038)

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Biochim. Biophys. Acta 1863, 40–9. DNA damage-induced apoptosis suppressor (DDIAS), a novel target of NFATc1, is associated with cisplatin resistance in lung cancer. 2016

Im, J.-Y., Lee, K.-W., Won, K.-J., Kim, B.-K, Ban, H.S., Yoon, S.-H., Lee, Y.-J., Kim, Y.-J., Song, K.-B. and Won, M.

Notes: The functional elements of the DDIAS promoter were deciphered with the Dual-Luciferase® Reporter Assay System utilizing constructs in pGL2 Basic and pRL-TK Control Vectors. NCI-H1703 cells were treated with siRNAs to either DDIAS, NFATc1 or a scrambled sequence. The cells with the scrambled sequence were unchanged, but the other two induced cytotoxicity and caspase-3/7 activation. Cytotoxicity was assessed with the CellTox™ Green Cytotoxicity Assay, and caspase-3/7 was assessed with a kinetic reagent. The two assays were visualized and quantified with an IncuCyte ZOOM System. (4707)

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Cell Signal 28, 284–93. Elongation factor 2 kinase promotes cell survival by inhibiting protein synthesis without inducing autophagy. 2016

Moore, C.E.J., Wang, X., Xie, J., Pickford, J., Barron, J., Regufe da Mota, S., Versele, M. and Proud, C.G.

Notes: Inhibition of HCT116 cell eEF2 kinase with the JAN-849 inhibitor lead to a dramatic increase in caspase-3/7 activity when the cells were cultured in medium without glucose for 24 hours. The increase could be blocked with cycloheximide (translation elongation inhibitor) or harringtonine (protein synthesis initiation inhibitor). Caspase activity was monitored with the Caspase-Glo® 3/7 Assay. A549 cells exposed to glucose-free medium had an increase in cytotoxicity that was partially abrogated by cycloheximide treatment. Knockdown of eEF2 kinase expression with siRNA treatment doubled the amount of cell death in glucose-free media, and cycloheximide treatment reduced cell death to about half. Cytotoxicity was monitored with the CellTox™ Green Cytotoxicity Assay using the end-point protocol. (4709)

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Biochim. Biophys. Acta 1860, 836–43. Glutamine drives glutathione synthesis and contributes to radiation sensitivity of A549 and H460 lung cancer cell lines. 2016

Sappington, D.R., Siegel, E.R., Hiatt, G., Desai, A., Penney, R.B., Jamshidi-Parsian, A., Griffin, R.J. and Boysen, G.

Notes: Both A549 and H460 cells were treated with the glutaminase inhibitor, BPTES. ATP content or viability was reduced about 30% in A549 cells and 60% in H460 cells. A549 and H460 cell ATP levels were mostly recovered by co-treatment with GSHE, a bioavailable form of glutathione. Despite a 60% decrease in ATP, H460 cells only demonstrated 5% cytotoxicity and A549 cells about 2.5% cytotoxicity. Co-treatment with GSHE reduced cytotoxicity to at or near background—no treatment levels. ATP content was assessed with the CellTiter-Glo® 2.0 Assay, and cytotoxicity was judged with the CellTox™ Green Cytotoxicity Assay. (4710)

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J. Lab. Autom. 21, 27–36. Precision cancer medicine in the acoustic dispensing Era: Ex vivo primary cell drug sensitivity testing. 2016

Kulesskiy, E., Saarela, J., Turunen, L. and Wennerberg, K.

Notes: The authors used the CellTiter-Glo® Cell Viability Assay and CellTox™ Green Cytotoxicity Assay to monitor the reproducibility of their acoustic dispensing. The authors report that adding CellTox™ Green Cytotoxicity Assay at plating "gives more solid signals than adding at the end of the assay” and speculate that the dye stabilizes the DNA of dead cells. The CellTiter-Glo® Assay data supports that the dye is nontoxic in the 72-hour incubation. The authors routinely predispense 12.5nl of CellTox™ Green with preplated compounds into 1536-well assay plates using an Echo 550 device and store plates under nitrogen gas in storage pods to produce assay-ready plates awaiting cell additions. The authors also report preliminary results of using the nonlytic RealTime-Glo® Viability Assay and find highly correlated results with the lytic CellTiter-Glo® Assay. The authors relate that use of the nonlytic RealTime-Glo® and CellTox™ Green Assays open the possibility of doing further work with the same cells. (4703)

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Oncogene , (Epub ahead of print). Topoisomerase IIα mediates TCF-dependent epithelial-mesenchymal transition in colon cancer. 2016

Zhou, Q., Abraham, A.D., Li, L., Babalmorad, A., Bagby, S., Arcaroli, J.J., Hansen, R.J., Valeriote, F.A., Gustafson, D.L., Schaack, J., Messersmith, W.A. and LaBarbera, D.V.

Notes: Topoisomerase IIα was identified as an important factor for metastasis of colorectal cancer involving the wnt signaling pathway. Microspheroids of SW620 cells, generated through ultra-low attachment plates, were used in the study. Activation of the wnt pathway was monitored with a reporter assay measured with the ONE-Glo™ Luciferase Assay System. Duplicate cells were monitored for viability with the CellTiter-Glo® 3D Cell Viability Assay. An ATP-competitive, N-terminal inhibitor of topoisomerase IIα was examined for cytotoxicity to the microspheroids. Cytotoxicity was monitored from 6–72 hours of exposure to the inhibitor using the CellTox™ Green Cytotoxicity Assay. Cytotoxicity increased over time with the inhibitor, and controls were essentially unaffected. (4708)

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PLos ONE 10, e0132309. Analysis of toxic amyloid fibril interactions at natively derived membranes by ellipsometry. 2015

Smith, R.A.S., Nabok, A., Blakeman, B.J.F., Xue, W.-F., Abell, B. and Smith, D.P.

Notes: Synthetic amyloid fibrils were created from α-synuclein and amyloid-β peptide 40. Fragment fibrils were made by freezing and sonicating the created fibrils. In vitro experiments suggested that fragment fibrils would interact and damage cellular membranes. This was confirmed by treating SH-SY5Y with both intact and fragmented fibrils over a 72-hour period. Cytotoxicity was measured with the CellTox™ Green Cytotoxicity Assay using the “zero step” addition to the cells at plating. Cytotoxicity was measured in the same wells at 24, 48 and 72 hours. (4714)

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Br. J. Cancer 112, 1536–45. Low-temperature plasma treatment induces DNA damage leading to necrotic cell death in primary prostate epithelial cells. 2015

Hirst, A.M., Simms, M.S., Mann, V.M., Maitland, N.J., O’Connell, D. and Frame, F.M.

Notes: Low-temperature atmospheric pressure plasmas (LTP) were examined for the mechanism of toxicity on cultured immortalized and primary prostate cancer cells. LTP induces an increase in H2O2 in the culture media as judged by the ROS-Glo™ H2O2 Assay leading to cell death as measured with the CellTox™ Green Cytotoxicity Assay. CellTox™ Green was quantified with a plate reader and verified by fluorescent microscopy. The cells experienced no caspase-3/7 activation as judged through use of the Caspase-Glo® 3/7 Assay, leading to the conclusion of necrotic cell death. (4711)

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Mol. Cell. Biol. 35, 1805–24. Molecular mechanism for the control of eukaryotic elongation factor 2 kinase by pH: Role in cancer cell survival. 2015

Xie, J., Mikolajek, H., Pigott, C.R., Hooper, K.J., Mellows, T., Moore, C.E., Mohammed, H., Werner, J.M., Thomas, G.J. and Proud, C.G.

Notes: A549 and HCT116 cells were transfected with an inducible shRNA to reduce eEF2K levels. Culturing of the cells at various medium pH lead to a decrease in viability and increase in cytotoxicity, which was increased when the shRNA was induced. Cell viability was measured with the CellTiter-Glo® Luminescent Cell Viability Assay, and cytotoxicity was measured with the CellTox™ Green Cytotoxicity Assay. (4712)

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Invest. New Drugs 33, 45–52. Novel benzoxazines as inhibitors of angiogenesis. 2015

Al-Rawi, S., Meehan-Andrews, T., Bradley, C. and Al-Rawi, J.

Notes: The authors were investigating benzoxazine analogs for potential anti-angiogenic activity through inhibition of the PI3K family of kinases. To eliminate highly toxic compounds, HUVEC cells were treated with 0–5µM of the test compounds and assessed for cytotoxicity at 6 hours and 24 hours using the CellTox™ Green Cytotoxicity Assay. (4704)

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Nat. Genet. 47, 1475–81. NRF2 regulates serine biosynthesis in non-small cell lung cancer. 2015

DeNicola, G.M, Chen, P.-H., Mullarky, D., Sudderth, J.A., Hu, Z., Wu, D., Tang, H., Xie, Y., Asara, J.M., Huffman, K.E., Wistuba, I.I., Minna, J.D., DeBerardinis, R.J. and Cantley, L.C.

Notes: Serine/Glycine biosynthesis was examined for a panel of 79 non-small cell lung carcinomas, and each could be classified into a high-synthetic and a low-synthetic group based on expression of four enzymes involved in serine/glycine synthesis from glucose. ATF4 was identified as a key mediator of NRF2 activity for serine/glycine biosynthesis through transcriptional activation and not regulation of ATF4 translation (the latter being examined in a dual-luciferase reporter assay using Dual-Glo® Luciferase Assay System). A byproduct of the conversion of serine to glycine is production of NADPH. Seven high-synthetic cell lines exhibited a marked decrease in NADPH levels with regard to NADP+ level when the rate-limiting serine/glycine synthetic enzyme was knocked down with shRNAs with little change in NADPH/NADP+ ratios in six low-synthetic cell lines. The levels of NADPH and NADP+ were monitored with the NADP/NADPH-Glo™ Assay. Expression of the various shRNAs used in the study did not induce cytotoxicity as judged by staining with the CellTox™ Green Cytotoxicity Assay and visual examination by fluorescent microscopy. The identity of all cell lines used in this study were verified by STR analysis with a PowerPlex® System. (4715)

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Mol. Cell. Biol. 35, 2385–99. RBM45 modulates the antioxidant response in amyotrophic lateral sclerosis through interactions with KEAP1. 2015

Bakkar, N., Kousari, A., Kovalik, T., Li, Y. and Bowser, R.

Notes: The role of RBM45 in the KEAP1/NRF2/ARE pathway was examined through transfection of HEK293 cells with either overexpression vectors or siRNAs. Overexpression of RBM45 increased ROS levels in cells with a concurrent increase in GSSG levels in comparison to GSH levels with a concurrent increase in cytotoxicity. Overexpression of RBM45 with a deleted nuclear localization signal slightly decreased the GSSG levels. The cytotoxic effect was abrogated with an siRNA directed to RBM45. GSH and GSSG levels were measured with the GSH/GSSG-Glo™ Assay, and cytotoxicity was measured with the CellTox™ Green Cytotoxicity Assay using the end-point protocol. (4713)

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PLOS Negl. Trop. Dis. 9, e0003484. Development and validation of a luminescence-base, medium throughput assay for drug screening in Schistosoma mansoni. 2015

Lalli, C. et al.

Notes: The authors demonstrated that CellTiter-Glo® Cell Viability Assay could be used for anthelminthic drug discovery.  The pilot study identified hits previously reported to have some anti-parasite activity.  To demonstrate that the CellTiter-Glo® Reagent was able to penetrate the schistosomula, the reagent was mixed with the CellTox™ Green Cytotoxicity Assay and visualized by fluorescent confocal microscopy.  (4648)

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Nat. Cell Biol. 17, 1556–68. Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.  2015

Tardito, S. et al.

Notes: Numerous cell lines were cultured for 72 hours in medium with glutamine, without glutamine or without glucose.  In all cell lines, glucose withdrawal was cytotoxic during the incubation.  The cells were highly tolerant of glutamine withdrawal with minimal cytotoxicity.  Cytotoxicity was measured with the CellTox™ Green Cytotoxicity Assay added at plating to cells in 12 well plates.  The fluorescence was measured every hour while incubated in an Essen BioScience IncuCyte FLR instrument. All cell lines used in the study were authenticated through STR analysis with the GenePrint® 10 System.  (4646)

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Cell Molec. Life Sci. 72, 367–81. Imaging oxygen in neural cell and tissue models by means of anionic cell-permeable phosphorescent nanoparticles.  2015

Dmitriev, R.I. et al.

Notes: Imaging oxygen in neural cell and tissue models by means of anionic cell-permeable phosphorescent nanoparticles. aCell permeable phosphorescent nanoparticle probes designed to enable the study of cell and tissue oxygenation were assessed for cytotoxicity in monolayer culture and rat brain slices.  The probe demonstrated no significant cytotoxicity on cultured PC12, mouse endothelial fibroblasts, primary cortical or cerebellar granule neurons in concentrations and exposure time tested as judged with the CellTiter-Glo® Cell Viability Assay.  Rat brain slices (400µm) were incubated with and without the nano probes and cytotoxicity visualized by fluorescent microscopy following 3 hour incubation with 0.05% CellTox™ Green Dye.  The probe had no effect on cell viability in the 3 to 24 hour time frame.  (4645)

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