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J. Virol. Methods 144, 86–90. A rapid DNA hybridization assay for the evaluation of antiviral compounds against Epstein-Barr virus. 2007

Prichard, M.N., Daily, S.L., Jefferson, G.M., Perry, A.L. and Kern, E.R.

Notes: The authors developed an assay to evaluate antiviral compounds for Epstein-Barr virus (EBV) infections. EBV DNA was isolated using the Wizard® SV 96 Genomic DNA Purification System, and 5µl was used for real-time PCR to quantify the viral DNA. Cytotoxicity of the antiviral compounds was assessed using treated, uninfected Akata cells compared to those with no treatment or infection. After three days when the viral DNA was harvested, cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay. (3761)

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Cancer Res. 67, 1254-1261. A small-molecule enhancer of signal transducer and activator of transcription 1 transcriptional activity accentuates the antiproliferative effects of IFN-γ in human cancer cells 2007

Lynch, R.A., Etchin, J., Battle, T.E. and Frank, D.A.

Notes: STAT1 is a transcription factor that is involved in a variety of cellular processes and behaves like a tumor suppressor in many ways. It is associated with apoptosis, inhibition of cyclin-dependent kinases, and it may mediate the antitumor effects of IFN-γ. The authors of this study designed a bioluminescent reporter assay to identify small molecules that enhance STAT1-dependent gene expression. The bioluminescent assay was performed in 384-well plates using both stably and transiently transfected cell lines. The screening assay was robust, producing a Z´factor value of 0.92, and it identified three compounds that specifically enhanced STAT1-dependent transcriptional activity. Firefly luciferase activity in stable cell lines was assessed using the Bright-Glo® Luciferase Assay System; luciferase activity in the transiently transfected cells was monitored using the Dual-Luciferase® Assay System. Viability of cells in culture was monitored using the CellTiter-Glo® Assay. (3732)

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Cancer Res. 67, 1472-1486. Adaptation of energy metabolism in breast cancer brain metastases. 2007

Chen, E.I., Hewel, J., Kreuger, J.S., Tiraby, C., Weber, M.R., Kralli, A., Becker, K., Yates, J.R., and Felding-Habermann, B.

Notes: This study investigated protein expression profiles in tumors from breast cancer brain metastases. Circulating tumor cells were isolated from a patient with stage IV breast cancer and injected into SCID mice. Tumor cells were then recovered from brain and bone lesions that subsequently developed in these mice. The protein expression profiles of the parent cell line were then compared with those from brain and bone tumors. More than 300 proteins that were up- or down-regulated in the brain tumor cells were identified. Classification of these proteins by function revealed that the majority were associated with cellular metabolism. Sixty-three differentially expressed proteins, including mainly cellular redox-active proteins and proteins involved in glucose and fatty acid oxidation, were selected for further study. Based on the expression data, a metabolic profile of brain metastatic cells was generated. Up-regulation of genes involved in oxidative energy metabolism as indicated by the proteomic analysis was confirmed by quantitative real-time PCR analysis. Consistent with the observation of increased glycolysis and oxidative phosphorylation, the authors also found that levels of cellular ATP were increased in cells from brain metastases. The CellTiter-Glo® Luminescent Cell Viability Assay was used to measure ATP levels in the primary, bone, and brain-derived tumor cells. The authors suggest that adaptation of the tumor cell energy metabolism is a key development in breast cancer brain metastasis. (3613)

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Assay Drug Dev. Technol. 5, 127–136. Bioluminescent assays for high-throughput screening 2007

Fan, F. and Wood, K.V.

Notes: The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays). (3737)

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J. Biomol. Scr. 12, 33. Development and validation of a high-throughput screen for inhibitors of SARS CoV and its application in screening of a 100,0000-compound library. 2007

Severson, W.E., Shindo, N., Sosa, M., Fletcher, T., White, L., Ananthan, S. and Jonsson, C.B.

Notes: The authors of this paper describe the validation of a bioluminescent assay using the CellTiter-Glo® Reagent to identify novel compounds that inhibit the cytopathic effect of SARS CoV. In this study, three cell viability assay reagents were evaluated: MTS, neutral red, and CellTiter-Glo® Reagent. The CellTiter-Glo®-based assay was chosen because it did not require washing or medium removal; it involved minimal pipetting steps and had a short incubation time, which reduced time in the BSL3 containment facility. The assay was used to screen 100,000 compounds and identified several compounds that inhibited CPE while having a minimal effect on cell viability. (3736)

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Proc. Natl. Acad. Sci. USA 104, 270-275. Identification of NVP-TAE684, a potent, selective and efficacious inhibitor of NPM-ALK 2007

Galkin, A.V., Melnick, J.S., Kim, S., Hood, T.L., Li, N., Li, L., Xia, G., Steensma, R., Chopiuk, G., Jiang, J., Wan, Y., Ding, P., Liu, Y., Sun, F., Schultz, P.G., Gray, N.S. and Warmuth, M.

Notes: NVP-TAE684 was identified as an inhibitor of the constitutive anaplastic lymphoma kinase (ALK) activity associated with the NPM-ALK fusion. NPM-ALK fusion protein is created by translocation event characteristic of anaplastic large-cell lymophomas. NVP-TAE684 was screened against a panel of 35 Ba/F3 cell lines expressing a variety of tyrosine kinases constitutively activated by fusion to TEL. The CellTiter-Glo® Luminescent Cell Viability Assay was used to detect any decrease in viability as a result of treatment with NVP-TAE684. The inhibitory effect of NVP-TAE684 was specific for ALK-associated cell proliferation. In a secondary screen to determine potency, TAE684 was screened against two human anaplastic large-cell lymphoma cell lines. Inhibition of proliferation correlated with dosage and was assessed using the Bright-Glo® Luciferase Assay System. (3738)

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J. Biomol. Scr. 12, 481–489. Identifying druglike inhibitors of myelin-reactive T cells by phenotypic high-throughput screening of a small-molecule library. 2007

Rossi, C., Padmanaban, D., Ni, J., Yeh, L-A., Glicksman, M.A. and Waldner, H.

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 (PLP139-151). 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)

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J. Biomol. Scr. 12, 546–559. Optimization procedure for small interfering RNA transfection in a 384-well format. 2007

Borawski, J., Lindeman, A., Buxton, F., Labow, M. and Gaither, L.A.

Notes: A lentiviral expression vector containing the firefly luciferase gene from a pGL3 Vector was transduced into SKOV3 cells in 384-well plates, transfected with various siRNAs and analyzed 72 hours later. The luciferase expression was determined using the Bright-Glo™ Luciferase Assay System and cell viability assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. (3729)

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J. Virol. 80, 130-7. "UnPAKing" Human Immunodeficiency Virus (HIV) replication: Using small interfering RNA screening to identify novel cofactors and elucidate the role of Group I PAKs in HIV infection 2006

Nguyen, D.G., Wolff, K.C., Yin, H., Caldwell, J.S. and Kuhen, K.L.

Notes: The CellTiter-Glo® Luminescent Cell Viability Assay was used to assess viability of HeLaCD4βgal or U373-Magi-CCR5E cells transfected with siRNAs that targeted potential proviral host factors for HIV infection. (3374)

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Proc. Natl. Acad. Sci. USA 103, 3153–3158. An efficient rapid system for profiling the cellular activities of molecular libraries 2006

Melnick, J.S., Janes, J., Kim, S., Chang, J.Y., Sipes, D.G., Gunderson, D., Jarnes, L., Matzen, J.T., Garcia, M.E., Hood, T.L., Beigi, R., Xia, G., Harig, R.A., Asatryan, H., Yan, S.F., Zhou, Y., Gu, X.J., Saadat, A., Zhou, V., King, F.J., Shaw, C.M., Su, A.I., Downs, R., Gray, N.S., Schultz, P.G., Warmuth, M. and Caldwell, J.S.

Notes: The CellTiter-Glo® Luminescent Cell Viability Assay was used for a cell-based kinase assay. The authors created 35 Ba/F3 cell lines transformed with constructs encoding tyrosine kinases fused to ETV6/Tel. Fusions of tyrosine kinases to ETV6/Tel generally have constitutive activity and can confer an IL-3-independent phenotype to Ba/F3 cells. Test compounds were assayed for inhibition of kinase activity and consequent decrease in cell viability. Cell viability was assessed using the CellTiter-Glo® Assay. The assays were performed in 1536 well plates using a completely automated robotic platform. (3394)

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Cancer Res. 66, 8109-8115. Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP-Ribose) polymerase inhibition. 2006

McCabe, N., Turner, N.C., Lord, C.J., Kluzek, K., Bialkowska, A., Swift, S., Giavara, S., O'Connor, M.J., Tutt, A.N., Zdzienicka, M.Z., Smith, G.C.M., and Ashworth, A.

Notes: The authors of this study investigated the basis of PARP inhibition sensitivity of cells containing mutations in BRCA1 or BRCA2 and whether the role of BRCA1 and 2 in homologous recombination might underlie the PARP inhibition sensitivity. They transfected HeLa cells with antisense constructs targeted against several proteins involved in homologous recombination and assessed sensitivity to PARP inhibition. Cells containing the RNAi constructs were treated with PARP inhibitors and blasticidin and cell viability was measured using the CellTiter-Glo® Cell Viability Assay. Antisense inhibition of several genes including those coding for RAD51, replication protein A1 and checkpoint kinase 2 among others, resulted in sensitivity to PARP inhibition. These results indicate that the role of BRCA1 and BRCA2 in homologous recombination contributes to the sensitivity to PARP inhibition. (3595)

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Br. J. Cancer 92, 1430–1441.. HGS-ETR1, a fully human TRAIL-receptor 1 monoclonal antibody, induces cell death in multiple tumor types in vitro and in vivo. 2005

Pukac, L., Kanakaraj, P., Humphreys, R., Alderson, R., Bloom, M., Sung, C., Riccobene, T., Johnson, R., Fiscella, M., Mahoney, A., Carrell, J., Boyd, E., Yao, X.T., Zhang, L., Zhong, L., von Kerczek, A., Shepard, L., Vaughan, T., Edwards, B., Dobson, C., Salcedo, T. and Albert, V.

Notes: The authors examine and characterize HGS-ETR1 (Mapatumumab), a fully human agonistic monoclonal antibody with high affinity and specificity for TRAIL-R1. HGS-ETR1 induced cell death in tumor cell lines was mediated by the activation of the extrinsic and intrinsic death signaling pathways. The tumor cell lines Colo205, HCT116, H460, H2122, ST486, SW480, RL95-2, WU.86.86, ES2, A498, WM793, SNU398, JURL-MKI and TTn were plated at 10,000 cells/well in 96-well white, opaque plates. The tumor cells were treated with the HGS-ETR1 or a isotope control antibody (ICmAB). After 48 hours, cell viability was determined by the CellTiter-Glo® Luminescent Cell Viability Assay. Luminescence was detected using a Northstar luminescent plate reader. Caspase 3/7 activity was assessed in the HGS-ETR1 and ICmAB treated tumor cells using the Apo-ONE™ Homogeneous Caspase-3/7 Assay. The cells were plated at 10,000 cells/well into black-walled 96-well plates and treated with the HGS-ETR1 and ICmAB for 6 hours at 37°C. Caspase activity was measured by reading the plates at 405nm using a fluorometric plate reader. (3314)

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Proc. Natl. Acad. Sci. USA 101(19), 7433-8. A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii. 2004

Carey, K.L., Westwood, N.J., Mitchison, T.J., Ward, G.E.

Notes: Toxoplasma gondii parasites and BS-C-1 cells were incubated for 60 minutes in Hanks' buffered saline solution (HBSS) containing either 100 µM small molecule being screened as potential invasion inhibitors or 0.25% DMSO. Cytotoxicity was determined using the CellTiter-Glo® Luminescent Cell Viability Assay. (3142)

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Proc. Natl. Acad. Sci. USA 101(24), 8969-74. Chemical genetics to identify NFAT inhibitors: potential of targeting calcium mobilization in immunosuppression 2004

Venkatesh, N., Feng, Y., DeDecker, B., Yacono, P., Golan, D., Mitchison, T., McKeon, F.

Notes: Primary T cells activated with anti-CD3 and anti-CD28 were treated with 2 µM of two calcium inhibitor compounds from a library of 16,000 plus cyclosporin A (CsA). The two compounds were able to reduce the apparent IC50 of CsA for the suppression of T cell activation without affecting the cell viability as measured using the CellTiter-Glo® Luminescent Cell Viability Assay. (3141)

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Mol. Biol. Cell 15(11), 5064-5074. Differential regulation of the TRAIL death receptors DR4 and DR5 by the signal recognition particle. 2004

Ren, Y.G., Wagner, K.W., Knee, D.A., Aza-Blanc, P., Nasoff, M. and Deveraux, Q.L.

Notes: Differences in the activation of apoptotic pathways by the TRAIL death receptors DR4 and DR5 were investigated. An siRNA screen was performed on HCT15 cells in 384-well plates to find genes that influence DR4- or DR5-mediated apoptosis differently. After induction of apoptosis using anti-DR4 or DR5 cross-linked antibodies, cell viability was assessed using the CellTiter-Glo® Reagent. It was found that the Signal Recognition Particle (SRP) plays a major role in DR4- but not DR5-mediated apoptosis. To further investigate this difference, stable HeLa cell lines were generated that expressed short hairpin RNA (shRNA) directed against SRP. The effects of various inducers of apoptosis were tested in these cell lines. Caspase activation was measured using the Caspase-Glo® 3/7 reagent. (3191)

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Clin. Can. Res. 10, 7547-7554. Epidermal growth factor receptor tyrosine kinase inhibition represses cyclin D1 in aerodigestive tract cancers.  2004

Petty, W.J., Dragnev, K.H., Memoli, V.A., Ma, Y., Desai, N.B., Biddle, A., Davis, T.H., Nugent, W.C., Memoli, N., Hamilton, M., Iwata, K.K., Rigas, J.R. and Dmitrovsky, E.

Notes: The CellTiter-Glo® Luminescent Cell Viability Assay was used to examine the proliferation of BEAS-2B (immortalized human bronchial epithelial (HBE)) cells after treatment with various concentrations of erlotinib, an inhibitor of the mitogenic effects of EGF. For these experiments, 3,000/well were seeded in 96-well plates and incubated for 72 hours with various concentrations of erlotinib. Other lung cell carcinoma cell lines (A549, H226, H358, and H441) were also tested for proliferation in the presence of erlotinib using the CellTiter-Glo® Assay system. The researchers used a luciferase construct that contained the cyclin D1 promoter to test the effects of erlotinib on cyclin d1 regulation. This construct, along with the pRL-TK vector, were co-transfected into BEAS-2B cells. Groups of cells were then treated with or without EGF and erlotinib and assayed for luciferase activity using the Dual-Luciferase® Reporter Assay System.  (3258)

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Cell Death Differ. 11, 1076–1083. Galectin-1β, a natural monomeric form of galectin-1 lacking its six amino-terminal residues promotes axonal regeneration but not cell death. 2004

Miura, T., Takahashi, M., Horie, H., Kurushima, H., Tsuchimoto, D., Sakumi, K. and Nakabeppu, Y.

Notes: In this study, the CellTiter-Glo® Luminescent Cell Viability Assay was used to analyze Jurkat cell viability after exposure to recombinant mouse galectin-1β (Gal-1β) or galectin-1α (Gal-1α). Data is presented as percent viable cells. Gal-1α was shown to have a more detrimental effect on cell viability.  (3172)

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Science 303, 832-835. Genome-wide RNAi analysis of growth and viability in Drosophila cells. 2004

Boutros, M., Kiger, A., Armknecht, S., Kerr, K., Hild, M., Koch, B., Haas, S., Heidelberg Fly Array Consortium, Paro, R., and Perrimon, N.

Notes: This paper describes use of RNA interference (RNAi) to screen the genome of Drosophila melanogaster for genes affecting cell growth and viability. 19,700 double stranded RNAs were used to screen approximately 91% of the genome. The screen consisted of transfection of approximately 12,000 Kc167 or S2R+ cells per well of a white 384-well plate with dsRNA. After a 5-day incubation, cell viability was assayed using the CellTiter-Glo® Luminescent Cell Viability Assay and a Molecular Dynamics Analyst HT. The authors report finding 438 target genes that affected cell growth or viability. (2843)

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J. Biol. Chem. 279, 5565-5572. Identification of cathepsin B as a mediator of neuronal death induced by Ab-activated microglial cells using a functional genomics approach. 2004

Gan, L., Ye, S., Chu, A., Anton, K., Yi, S., Vincent, V.A., von Schack, D., Chin, D., Murray, J., Lohr, S., Patthy, L., Gonzalez-Zulueta, M., Nikolich, K., and Urfer, R.

Notes: The authors describe use of the CellTiter-Glo® Luminescent Cell Viability Assay to assess viability of rat primary cortical neurons after treatment with Aβ42 peptide or Aβ42 peptide-BV2 murine microglial cell conditioned media. The CellTiter-Glo® Luminescent Cell Viability Assay was also used to assess cell viability during Aβ42 peptide treatment of BV2 cells transfected with siRNAs targeting sequences in cathepsin B, cathepsin L, TIMP2, and AIF1 genes. (2842)

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Cancer Res. 64(13), 4487-97. Increased susceptibility of poly(ADP-ribose) polymerase-1 knockout cells to antitumor triazoloacridone C-1305 is associated with permanent G2 cell cycle arrest. 2004

Wesierska-Gadek, J., Schloffer, D., Gueorguieva, M., Uhl, M., Skladanowski, A.

Notes: The antiproliferative drug C-1305 was added at 0.1-2 µM or amsacrine at 0.1-1 µM for 24 hours, and then the cells were cultivated for an additional 48 hours in a drug-free medium. Cell proliferation was determined using the CellTiter-Glo® Luminescent Cell Viability Assay. (3140)

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Assay Drug Dev. Technol. 2, 51-62. Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. 2004

Riss, T.L. and Moravec, R.A.

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)

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J. Virol. 77(1), 624-630. Disruption of CCL21-induced chemotaxis in vitro and in vivo by M3, a chemokine-binding protein encoded by murine gammaherpesvirus 68. 2003

Jensen, K.K., Chen, S.C., Hipkin, R.W., Wiekowski, M.T., Schwarz, M.A., Chou, C.C., Simas, J.P., Alcami, A. and Lira, S.A.

Notes: The M3 gene product of gammaherpesvirus 68 (MHV-68) has been shown to bind certain chemokines and to block some chemokine signaling in vitro. This study demonstrates that M3 blocks in vitro chemotaxis induced by CCL19 and CCL21. Approximately 30,000 Ba/F3-hCCR7 cells were used in a chemotaxis assay. After a 2-hour incubation at 37° in a 5% CO2 atmosphere, cell migration was quantitated using the CellTiter-Glo™ Luminescent Cell Viability Assay. The number of migrating cells was determined via interpolation from standard curves.  (2702)

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Biol. Pharm. Bull. 26(2), 233-240. Effect of CAWS, a mannoprotein-beta-glucan complex of Candida albicans, on leukocyte, endothelial cell, and platelet functions in vitro. 2003

Kurihara, K., Shingo, Y., Miura, N.N., Horie, S., Usui, Y., Adachi, Y., Yadomae, T., Ohno, N.

Notes: The in vivo effect of the water-soluble polysaccharide fraction (CAWS) produced by Candida albicans was studied.  It is found that at high doses CAWS inhibited proliferation of spleenocytes and inhibited growth rate of cultured monophages (RAW264.7) in a dose dependent manner.  To test cell viability of RAW264.7, cells were plated RPMI1640 with gentmycin sulfate and 10% FCS to 5x104 or 1x105 cells/mL, allowed to attach to the dish for two hours, then incubated for 24 hours at 37° with 5% CO2 in the presence of CAWS or sonifilan.  The cells were then counted or assayed for viability using CellTiter-Glo™. (2711)

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Cancer Res. 63(18), 5866-73. Endothelial precursor cells as a model of tumor endothelium: characterization and comparison with mature endothelial cells. 2003

Bagley, R.G., Walter-Yohrling, J., Cao, X., Weber, W., Simons, B., Cook, B.P., Chartrand, S.D., Wang, C., Madden, S.L., Teicher, B.A.

Notes: The generation times of the EPC, HUVEC, and HMVEC cells were determined over a 4-day period. Cells were plated in a 96-well plate format at 2000 or 3000 cells/well in triplicate and were assayed daily using the CellTiter-Glo® Luminescent Cell Viability Assay. (3143)

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Science 301(5636), 1099-102. Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation. 2003

Gebert, B., Fischer, W., Weiss, E., Hoffmann, R., Haas, R.

Notes: The CellTiter-Glo® Luminescent Cell Viability Assay was used to assess the proliferation of both Jurkat cells and peripheral blood lymphocytes (PBLC) treated with various strains of Helicobacter pylori and isogenic Campylobacter jejuni or Escherichia coli HB101. In addition, the proliferation of PBLCs and Jurkat cells was assayed after treatment with concentrated bacterial culture supernatant with or without vacuolating cytotoxin VacA. (3144)

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