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5, 163–71. Barley as a green factory for the production of functional Flt3 ligand. 2010

Erlendsson, L.S., Muench, M.O., Hellman, U., Hrafnkelsdóttir, S.M., Jonsson, A., Balmer, Y., Mäntylä, E. and Orvar, B.L.

Notes: The authors explore barley (Hordeum vulgare) as a means of expressing recombinant human Flt3 ligand, which is a growth factor involved in proliferation and differentiation of stem cells and development of various immune cells. As part of their quality control, they performed in-gel proteolytic digestion and mass spectrometry. The recombinant Flt3 ligand was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Coomassie® blue staining, excision of the protein band, destaining, drying of the gel slice and digestion with Sequencing Grade Modified Trypsin at 30°C overnight prior to mass spectrometry. To test biological activity, the authors treated human acute myeloid leukemia cells with Flt3 expressed in barley or a commercial source of Flt3 then measured cell proliferation using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (4352)

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Cancer Res. 69, 272–281. Cyclin-dependent kinase-3-mediated c-Jun phosphorylation at Ser63 and Ser73 enhances cell transformation. 2009

Cho, Y.Y., Tang, F., Yao, K., Lu, C., Zhu, F., Zheng, D., Pugliese, A., Bode, A.M. and Dong, Z.

Notes: This paper examined the role of the Cdk3/c-Jun signaling in EGF-stimulated cell proliferation and cell transformation. An AP-1 luciferase reporter plasmid construct was contransfected with various expression vector combinations of Cdk3, Cdk2, c-Jun, c-Jun M63/73, Cdk3-DN and cyclin C and the phRL-SV40 Vector as a normalization control. Cells were lysed at room temperature for 30 minutes with gentle shaking and luciferase activity measured using the Dual-Luciferase® Reporter Assay System. SaoS-2 cells transfected with a mock and Cdk3 RNAi vector were seeded at a density of 4 x 103 in 96-well plates with 20µl of medium. After 24 hours, 20µl of CellTiter® 96 AQueous One Solution was dispensed to each well, and the plate incubated for 1 hour at 37°C. The reaction was stopped using 25µl of 10% SDS and absorbance was measured at 492 and 690nm. For the CheckMate™ Mammalian Two-Hybrid System, the plasmid constructs, pACT-c-Jun, pBIND-Cdk3 and pG5luc, were cotransfected into HEK293 cells at no more than 100ng/well in a 48-well plate. After incubation and lysis, 20µl of lysate was dispensed into each well of a 96-well luminescence plate. Firefly and Renilla luciferase activity was detected. (4028)

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Nucl. Acids Res. 37, 78–95. Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage. 2009

Wilson, P.M., Fazzone, W., LaBonte, M.J., Lenz, H.J. and Ladner, R.D.

Notes: The authors examined the role of p53 in modulating dUTPase promoter activity. Base substitution mutations of Sp1- and E2F-binding sites in the dUTPase promoter were performed using the GeneEditor™ in vitro Site-Directed Mutagenesis System. Each mutant was confirmed by DNA sequencing. To determine growth inhibition, HCT116 human colon cancer cells were seeded in 96-well plates at 3 × 103 cells/well and treated with 5-fluorouracil (5-FU), fluorodeoxyuridine (FUdR), oxaliplatin or in combination. After 72 hours, the CellTiter® 96 AQueous One Solution was dispensed into each well and absorbance measured. RNA was isolated from HCT116 p53+/+ and HCT116 p53-/- cells. cDNA was reverse transcribed from 200ng total RNA followed by multiplex qPCR using the Plexor™ qPCR System to amplify dUTPase, thymidylate synthase and GAPDH, a housekeeping gene. The 1.2 kb region of the dUTPase promoter upstream of the transcriptional start site was amplified by PCR and the fragment cloned into the pGL3-Basic Vector. Truncated promoters were also generated by PCR and cloned into the same vector. Drosophila SL-2 cells and HCT116 cell lines were seeded in a 24-well plate and transfected with dUTPase pGL3 promoter constructs or with pCI-Neo:p53WT, pCI-Neo:p53MUT and the empty pCI-neo Mammalian Expression Vector; all transfections included the pRL-TK Vector at a ratio of 1:10. After six hours, the cells were incubated in either fresh medium or medium containing a cytotoxic agent at the appropriate concentration. Thirty hours later, the cells were lysed, quantitated by Western blotting and 20µl of lysate analyzed with the Dual-Luciferase® Reporter Assay System. Electrophoretic mobility shift analyses (EMSA) were performed using –64 to –91 of the dUTPase-nuclear isoform transcriptional start site in the Gel Shift Assay System. (4031)

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Cancer Res. 68, 7650–7660. Cyclin-dependent kinase 3-mediated activating transcription factor 1 phosphorylation enhances cell transformation. 2008

Zheng, D., Cho, Y.Y., Lau, A.T., Zhang, J., Ma, W.Y., Bode, A.M. and Dong, Z.

Notes: To examine the role of cyclin-dependent kinase (cdk)-3 expression in cancer cell lines, potential targets of cdk3 phosphorylation were examined. The CheckMate™ Mammalian Two-Hybrid System was used to test for transcription factor binding partners of cdk in HEK293 cells. Cell proliferation of T96G cells stably transfected with either cdk3 or vector was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3983)

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Cancer Res. 68, 6803-6809. Mutation of genes affecting the RAS pathway is common in childhood acute lymphoblastic leukemia 2008

Case, M., Matheson, E., Minto, L., Hassan, R., Harrison, C.J., Bown, N., Bailey, S., Vormoor, J., Hall, A.G. and Irving, J.A.E.

Notes: The authors of this study investigated the relationship of somatic mutations that deregulate the RAS-RAF-MEK-ERK pathway and Acute Lymphoblastic Leukemia (ALL) and its progression to relapse in some patients. They show that such mutations are common in ALL and its recurrence. Furthermore, lymphoblasts from patients with mutations that were associated with upregulation of ERK showed increased cytotoxicity over wild-type controls when treated with U0126, suggesting that specific ERK inhibitors may eventually yield useful therapeutics. Following drug exposure, cytotoxic effects were assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3905)

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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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Mol. Cell. Biol. 26, 6412-6424. Heregulin-dependent delay in mitotic progression requires HER4 and BRCA1. 2007

Muraoka-Cook, R.S., Caskey, L.S., Sandahl, M.A., Hunter, D.M., Husted, C., Strunk, K.E., Sartor, C.I., Rearick, W.A., McCall, W., Sgagias, M.K., Cowan, K.H., and Earp, H. S.

Notes: Heregulin is a growth factor that binds to the HER/ErbB family of receptors, which includes four HER members. HER1 and HER2 overexpression in breast cancer cells is associated with higher malignancy and poorer prognosis. However, HER4 expression is associated with longer survival and more positive prognosis. The authors of this study investigated HER4-dependent growth inhibition that is mediated by heregulin. The authors determined the absolute levels of HER4 mRNA in several human breast cancer and mouse cancer cell lines. For this determination, relative cell numbers were determined using a CellTiter® AQueous MTS assay. Apoptosis in heregulin-treated or untreated cells was also determined using the DeadEnd™ Colorimetric TUNEL System. The authors concluded that heregulin decreases growth of HER4-positive breast cancer cells, and that this growth inhibition is dependent on BRCA1. (3596)

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J. Biol. Chem. 281, 22656–22664. The SWI/SNF chromatin remodeling subunit BAF57 is a critical regulator of estrogen receptor function in breast cancer cells. 2007

Garcia-Pedrero, J.M, Kiskinis, E., Parker, M.G., and Belandia, B.

Notes: To examine the role that BAF57, a transcriptional modulator of the estrogen receptor (ER), may have in breast cancer, BT549 cells were transfected with a reporter vector (pGL3-Basic with two estrogen response elements and the human pS2 promoter), the control pRL-CMV Vector and combinations of the following expression vectors: mERα or hERβ, BAF57, SRC1e, SRC1a and RAC3. After 16 hours, the cells were treated with 17β-estradiol. Twenty-four hours later, the cells were harvested and the luciferase activities assayed using the Dual-Luciferase® Reporter Assay System. GST-BAF57 (full-length, N- or C-domain) fusion protein was bound to Sepharose beads and incubated with 17β-estradiol or vehicle and wildtype or one of various mERα interaction domain mutants, which have been expressed and labeled with 35S methionine using a TNT® rabbit reticulocyte lysate system. The beads were washed and analyzed for bound protein. ZR-75-1 cells were transfected with BAF57 siRNA then treated with 17β-estradiol 24 hours later. Cell proliferation was measured using the CellTiter® 96 AQueous One Solution Cell Proliferation Assay. (3599)

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J. Biol. Chem. 282, 13059-13072. XAF1 mediates tumor necrosis factor-α-induced apoptosis and X-linked inhibitor of apoptosis cleavage by acting through the mitochondrial pathway 2007

Straszewshi-Chavez, A., Visintin, I.P., Karassina, N., Los, G., Liston, P., Halaban, R., Fadiel, A. and Mor, G.

Notes: The authors sought to determine the mechanism by which first-trimester trophoblasts resist FAS ligand-induced apoptosis but remain sensitive to TNFα-mediated apoptosis. First trimester trophoblasts express XAF1 [X-linked inhibitor of apoptosis (XIAP)-associated factor 1], which may be involved in regulating their response to proapoptotic signals. The authors created HaloTag™-XAF1 fusion constructs and transiently transfected the first trimester trophoblast cell line (3A). Cells were labeled with the HaloTag™ TMR ligand, and XAF1 was shown to localize to the cytoplasm. 3A cells transiently transfected with the fusion construct were also separated into cytoplasmic and mitochondrial fractions. The fractions were labeled with HaloTag™ TMR ligand. Expression of the fusion peaked at 48 hours after transfection in both mitochondrial and cytoplasmic fractions. TNFα-treatment of 3A cells induced translocation of endogenous XAF1 to the mitochondria. The authors used the Caspase-Glo® Assays to demonstrate activation of caspase-3 and caspase-9 in response to expression of XAF-1. They also show that caspase-3 activation and XIAP cleavage correlate with translocation of endogenous XAF1 to mitochondria. Viability of 3A and primary trophoblasts over expressing XAF1 was evaluated using the CellTiter® 96 AQueous One-Solution Assay. (3760)

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J. Biol. Chem. 281, 14700–14710. Attenuation of peroxisome proliferator-activated receptor gamma (PPARgamma) mediates gastrin-stimulated colorectal cancer cell proliferation. 2006

Chang, A.J., Song, D.H. and Wolfe, M.M.

Notes: The researchers sought to determine whether PPARγ expression might direct the effects of gastrin in proliferation of colorectal cancer cells (CRC). They determined that cell line DLD-1 cells had both PPARγ and gastrin receptors. They demonstrated that gastrin stimulated CRC cell proliferation with a coincident decrease in PPARγ levels. These studies show that gastrins trophic properties could be due in part to transactivation of EGFR and phosphorylation of ERK1/2, leading to a decrease in PPARγ activation.

The authors used CellTiter 96® Aqueous One Solution Cell Proliferation Assay to measure cell growth of the CRC cell line DLD-1.

The DLD-1 cells were transiently transfected with a luciferase vector, and FuGENE® 6 Transfection Reagent was used at a DNA ratio of 3:1 in 24-well plates. To normalize for transfection efficiency, the cells were co-transfected with a β-Gal reporter construct. The Dual-Luciferase® Assay System was used to measure PPARgamma activity with values then normalized to Beta-Gal, measured with the β-Galactosidase Enzyme Assay System.  (4280)

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Clin. Can. Res. 11, 2345-2354. 4-Hydroxytamoxifen inhibits proliferation of multiple myeloma cells in vitro
through down-regulation of c-Myc, up-regulation of p27Kip1, and modulation of
Bcl-2 family members.
2005

Gauduchon, J., Gouilleux, F., Maillard, S., Marsaud, V., Renoir, J.M., and Sola, B.

Notes: Uses CellTiter 96 Aqueous One Solution for cell viability determination (3313)

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Cancer Res. 65, 9727–34. Cyclin E overexpression obstructs infiltrative behavior in breast cancer: a novel role reflected in the growth pattern of medullary breast cancers. 2005

Berglund, P., Stighall, M., Jirstrom, K., Borgquist, S., Sjolander, A., Hedenfalk, I. and Landberg, G.

Notes: To examine the influence of cyclin E overexpression on gene expression in breast cancer, the breast cancer cell line MDA-MB-468 was stably transfected with a cyclin E-GFP fusion construct or GFP alone. RNA from two of these clones was isolated and pooled prior to reverse transcription and labeling. The microarrays were filtered and used in GoMiner analysis. Attachment assays were also performed with the stable cell lines. In these assays, the cells were allowed to adhere to 96-well plates for 1 hour, washed with PBS and then incubated with the CellTiter 96® AQueous One Solution Cell Proliferation Assay for 1.5 hours. Absorbance was used to measure the number of attached cells. (3388)

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Cancer Res. 65, 4500-4505. In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 against clinically relevant Imatinib-resistant Abl kinase domain mutants. 2005

O'Hare, T., Walters, D.K., Stoffregen, E.P., Jia, T., Manley, P.W., Mestan, J., Cowan-Jacob, S.W., Lee, F.Y., Heinrich, M.C., Deininger, M.W.N. and Druker, B.J.

Notes: Ba/F3 cells were transfected with constructs encoding either wildtype or mutant (Imatinib-resistant) Bcr-Abl kinase. Src-family kinase activity was assayed using the SignaTECT® Protein Tyrosine Kinase Peptide 2 and SAM Biotin Capture Membranes. Cell viability was measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3405)

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Genes Dev. 18(9), 1060-71. Angiopoietin-1 modulates endothelial cell function and gene expression via the transcription factor FKHR (FOXO1) 2004

Daly, C., Wong, V., Burova, E., Wei, Y., Zabski, S., Griffiths, J., Lai, K.M., Lin, H.C., Ioffe, E., Yancopoulos, G.D., Rudge, J.S.

Notes: HUVECs were infected with adenoviruses encoding either GFP, forkhead transcription factor (FKHR), or FKHR-TM for 24, 32, or 45 hours. At each time point, the relative number of living cells was determined by the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3155)

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Appl. Environ. Microbiol. 70(5), 2567-76. Characterization of humanized antibodies secreted by Aspergillus niger. 2004

Ward, M., Lin, C., Victoria, D.C., Fox, B.P., Fox, J.A., Wong, D.L., Meerman, H.J., Pucci, J.P., Fong, R.B., Heng, M.H., Tsurushita, N., Gieswein, C., Park, M., Wang, H.

Notes: Aliquots of 1.8 x 103 cells were placed in 96-well microtiter plates and the cells were allowed to adhere for 6 hours before medium containing two-fold serial dilutions of Aspergillus niger-derived antibody from 20 to 0.078 g/ml (final concentrations of 10 to 0.039 g/ml) or medium alone then were added to the wells. After 72 hours incubation, relative proliferation was measured by using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (3153)

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Am. J. Physiol. Lung Cell. Mol. Physiol. 286(4), L866-76. Effects of sham air and cigarette smoke on A549 lung cells: implications for iron-mediated oxidative damage. 2004

Mayo, J.J., Kohlhepp, P., Zhang, D., Winzerling, J.J.

Notes: A549 cells were grown to 80% confluence before being exposed to smoke or sham air for 4, 6 or 22 hours. Cells were harvested by trypsinization and viability was determined using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3165)

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Cancer Res. 64(13), 4637-47. Prolonged extracellular signal-regulated kinase 1/2 activation during fibroblast growth factor 1- or heregulin beta1-induced antiestrogen-resistant growth of breast cancer cells is resistant to mitogen-activated protein/extracellular regulated kinase kinase inhibitors. 2004

Thottassery, J.V., Sun, Y., Westbrook, L., Rentz, S.S., Manuvakhova, M., Qu, Z., Samuel, S., Upshaw, R., Cunningham, A., Kern, F.G.

Notes: Subconfluent ML20 cells were stripped of estrogens over three days then plated in a 96-well plate at 5000 cells/well. After overnight attachment, the cells were treated with fresh media containing 5% FBS plus ICI 182780 with or without each of the following: heregulin, epidermal growth factor; fibroblast growth factor 1 and β-estradiol or each of the factors individually. Under these various media conditions, the cells were treated with U0126. After five days, the cells were assayed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3154)

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Biomaterials 25, 843–850. Protection of insulin secreting cells from nitric oxide induced cellular damage by crosslinked hemoglobin. 2004

Chaea, S.Y., Leeb, M., Kimb, S.W. and Baeb, Y.H.

Notes: The authors studied the effect of nitric oxide stress on rat Islets of Langerhans cells and rat insulinoma cell line (RINm5F) and to determine if cross-linked hemoglobin (Hb-C)could mediate the stress and subsequent apoptosis.  The rat cells were treated with a nitric oxide donor SNAP and the DeadEnd™ Colorimetric TUNEL System was used to measure the effect of NO with or without Hb-C.  In addition, the cell viability was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay.  Twenty-four hours after SNAP treatment, the level of NO2 (a by-product of NO) was measured using Griess Reagent to determine the level of nitric oxide production induced. (3053)

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J. Physiol. 558(Pt 1), 181-91. Response of human cells to desiccation: comparison with hyperosmotic stress response. 2004

Huang, Z., Tunnacliffe, A.

Notes: Cells were plated in a 96-well plate and incubated until near confluence. The cells were subsequently dried by removing the media before being placed in a humid atmosphere for 2-24 hours. Then the cells were rehydrated by incubating in media for two hours before being assayed for cell viability using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3163)

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Mol. Cell. Biol. 24(7), 2853-62. Silencing of chromatin assembly factor 1 in human cells leads to cell death and loss of chromatin assembly during DNA synthesis. 2004

Nabatiyan, A., Krude, T.

Notes: HeLa cells were grown in 24-well plates and transfected with nontarget and p60 siRNAs with untransfected cells as a control. After 24 and 48 hours, loss of cell viability upon silencing of p60 was assessed by incubating the cells with the CellTiter 96® AQueous One Solution Cell Proliferation Assay for five minutes and then stopping the reaction. (3156)

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J. Invest. Dermatol. 123(2), 380-7. The novel synthetic oleanane triterpenoid CDDO (2-cyano-3, 12-dioxoolean-1, 9-dien-28-oic acid) induces apoptosis in Mycosis fungoides/Sezary syndrome cells. 2004

Zhang, C., Ni, X., Konopleva, M., Andreeff, M., Duvic, M.

Notes: Aliquots of 5 x 104 cells/well were distributed in 96-well plates and incubated with 1, 2, and 5 µM novel synthetic oleanane triterpenoid CDDO for 48 hours. Then the relative cell viability was determined by comparing untreated control (DMSO) to treated groups using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3164)

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Clin. Can. Res. 10(6), 1901-10. Transendothelial migration of myeloma cells is increased by tumor necrosis factor (TNF)-alpha via TNF receptor 2 and autocrine up-regulation of MCP-1. 2004

Johrer, K., Janke, K., Krugmann, J., Fiegl, M., Greil, R.

Notes: 0.5 x 106 cells/ml were seeded in 96-well plates and incubated for 24 or 48 hours with or without recombinant human TNF-α or recombinant human MCP-1. The CellTiter 96® AQueous One Solution Cell Proliferation Assay was added to the cells and incubated for 4 hours before the absorbance was measured. (3157)

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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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Clin. Can. Res. 9, 2933-2939. Expression of constitutively active Akt-3 in MCF-7 breast cancer cells reverses the estrogen and tamoxifen repsonsivity of these cells in vivo 2003

Faridi, J., Wang, L., Endermann, G., Roth, R.A.

Notes: MCF-7 cells were transfected with a plasmid encoding consitutitively active Akt-3. To assess estrogen-related transcriptional activity, the cells were transfected with an ERE-luciferase (firefly) reporter and a Renilla luciferase control plasmid. Dual  luciferase assays were performed to determine the effect of estrogen on transcription in these Akt-3 expressing cells.  Additionally, proliferation assays were performed using the CellTiter® AQueous One Solution Cell Proliferation Assay and showed that the Akt-3 expression confers serum-independent growth on the cells. (2709)

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Assay Drug Dev. Technol. 3, 469-477. HTS Compatible Assay for Antioxidative Agents Using Primary Cultured Hepatocytes 2003

Gaunitz, F. and Heise, K.

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)

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