Optimization of 3D culture cell proliferation - part 4

Three major variables impact the 3D culture proliferation assay: limitations of detection, cell seeding density, and duration of 3D culture. While most established 3D culture protocols should be compatible with this assay, it may be helpful to empirically test and optimize these conditions before commencing pharmacological screening to promote assay sensitivity and reproducibility. This starts by developing a standard curve to determine the limitations for detection for each cell line. Cells should be serially diluted and seeded on ECM-coated stripwells before evaluating each well using the 3D culture cell proliferation assay. Absorbance should be evaluated at 1-, 2-, 3-, and 4-hr time points after adding substrate to determine the optimal incubation period. Background values are subtracted, data averaged, and standard curves plotted. The optimal curve is linear and has the lowest standard deviations. The standard curve will indicate what the highest number of detectable cells are based on the largest value within the linear range; it will also specify the lowest number of detectable cells in each well. These values provide the dynamic range for the assay. Since the ECM may affect cell proliferation rates, a growth curve may also be helpful in determining the optimal duration for 3D culture. Here, seeding densities may be used from an existing model or determined from the standard curve. When making a determination from the standard curve, sensitivity of this assay depends on the number of population doublings and overall absorbance, as a consequence of cell number. In the figure below, the seeding concentration for MDA-MB-231 cells in 3D BME culture is determined to allow for a 20-fold increase in cell number, which is just over five population doublings. The maximum value for the linear standard curve, 100,000 cells, is divided by 20 to give a seeding concentration of 5000 cells per well. Here, the cell doubling rate starts around two days and appears to increase with time, reaching saturation around day six. Since the MDA-MB-231 cells have a doubling rate of approximately 24 hr in traditional 2D cell culture, this represents an initial decrease in proliferation in the 3D BME culture model.

References
1. Birgersdotter, A.; Sandberg, R.; Ernberg, I. Gene expression perturbation in vitro—a growing case for three-dimensional (3-D) culture systems. Semin. Cancer Biol. 2005, 15(5), 405–12.
2. Cukierman, E.; Pankov, R.; Yamada, K.M. Cell interactions with three-dimensional matrices. Curr. Opin. Cell Biol. 2002, 14(5), 633–40.
3. Nelson, C.M.; Bissell, M.J. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Ann. Rev. Cell Dev. Biol. 2006, 22, 287–309.
4. Schuetz, E.G.; Li, D.; Omiecinski, C.J.; Muller-Eberhard, U.; Kleinman, H.K.; Elswick, B.; Guzelian, P.S. Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix. J. Cell Physiol. 1988, 134(3), 309–23.
5. Barcellos-Hoff, M.H.; Aggeler, J.; Ram, T.G.; Bissell, M.J. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development 1989, 105(2), 223–35.
6. Webber, M.M.; Bello, D.; Kleinman, H.K.; Hoffman, M.P. Acinar differentiation by non-malignant immortalized human prostatic epithelial cells and its loss by malignant cells. Carcinogenesis 1997, 18(6), 1225–31.
7. Grant, D.S.; Tashiro, K.; Segui-Real, B.; Yamada, Y.; Martin, G.R.; Kleinman, H.K. Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro. Cell 1989, 58(5), 933–43.
8. Griffith, L.G.; Swartz, M.A. Capturing complex 3-D tissue physiology in vitro. Nat. Rev. Mol. Cell Biol. 2006, 7(3), 211–24.
9. Petersen, O.W.; Ronnov-Jessen, L.; Howlett, A.R.; Bissell, M.J. Interaction with Basement Membrane Serves to Rapidly Distinguish Growth and Differentiation Pattern of Normal and Malignant Human Breast Epithelial Cells. Proceedings of the National Academy of Sciences 1992, 89(19), 9064–8.
10. Fridman, R.; Giaccone, G.; Kanemoto, T.; Martin, G.R.; Gazdar, A.F.; Mulshine, J.L. Reconstituted Basement Membrane (Matrigel) and Laminin Can Enhance the Tumorigenicity and the Drug Resistance of Small Cell Lung Cancer Cell Lines. Proceedings of the National Academy of Sciences 1990, 87(17), 6698–6702.
11. Hodkinson, P.S.; Elliott, T.; Wong, W.S.; Rintoul, R.C.; Mackinnon, A.C.; Haslett, C.; Sethi, T. ECM overrides DNA damage-induced cell cycle arrest and apoptosis in small-cell lung cancer cells through beta1 integrin-dependent activation of PI3-kinase. Cell Death Differ. 2006, 13(10), 1776–88.
12. Pogany, G.; Timar, F.; Olah, J.; Harisi, R.; Polony, G.; Paku, S.; Bocsi, J.; Jeney, A.; Laurie, G.W. Role of the basement membrane in tumor cell dormancy and cytotoxic resistance. Oncology 2001, 60(3), 274–81.

Series brought to you by Trevigen, Inc.

Assessing 3D culture cell proliferation - part 3

The ability of cells to remain viable and proliferate in a physiological environment is one of the most direct measures of cell health, and alterations in these proliferation rates suggest changes to biological processes within the cell. Since these values correlate to the number of cells present at the
time of assay, the data relate to changes in processes governed by cell cycle or cell death. As it is, decreasing the proliferation rate is generally correlated negatively to cell health where there is either a decrease in cell cycle progression or an increase in cell death. Likewise, dramatic increases in cell proliferation rates may be associated with loss of cell cycle control and an increase in disease pathogenesis.

The adaptation of 3D culture for studying cellular proliferation demonstrates that the ECM may impact the pharmacological dose response, and by evaluating cells in the proper context, these models elucidate the potential for drug resistance and accelerate translational research. Neoplastic cells in 3D culture have demonstrated drug resistance to many chemotherapeutic agents including doxorubicin, etoposide, cisplatin, and the nitrogen mustard, (10) and these cultures have been shown to promote resistance to ionizing and ultraviolet radiation as well. (11, 12)
Since cell–matrix interactions are mediated via cell surface receptors, know as integrins, and their subsequent signaling cascades, the search for targets that can abrogate the protective effects of the ECM and sensitize cancers to existing treatment therapies has begun, and these assays will be instrumental in identifying these targets and screening therapeutic compounds. During this process, stripwells from a 96-well plate are coated with ECM proteins which form hydrogels that mimic the tissue microenvironment. When cells are added to these wells, specific interactions between the cell and the ECM proteins induce a change in the cells’ biological program, and as the cells proliferate, they assume structural and functional characteristics as seen in vivo. Figure 1 demonstrates differences in cell morphology and structure formation for normal, MCF-10A, and malignant, MDA-MB-231, mammary epithelial cells cultured in 2D and 3D cultures. The MCF-10A cells grow in discrete cobblestone monolayers on plastic, and form glandular structures when cultured on BME; likewise, the MDA-MB-231 cells grow in spindle-shaped monolayers on plastic and form disorganized masses when cultured on BME. Cells may be monitored via microscopy during the course of the assay to evaluate cell morphology; structure formation; and, to some extent, viability. At the end of the culture, the 3D culture cell proliferation reagent is added to each well, and the viable cells within the well convert the substrate to a yellow product that absorbs at 450 nm. The absorbance can be evaluated in a 96-well plate reader, and the relative proliferation can be determined based on these absorbance values.

Figure 1. 3D cultures promote cell assembly as seen in vivo. The normal mammary epithelial cell line, MCF-10A, grows as a cobblestone monolayer on plastic (a) and forms glandular structures in a 3D BME culture (b). The malignant mammary epithelial cell line, MDAMB-231, grows as spindle-shaped monolayers on plastic (c) and forms a disorganized mass in a 3D BME culture (d).

References
1. Birgersdotter, A.; Sandberg, R.; Ernberg, I. Gene expression perturbation in vitro—a growing case for three-dimensional (3-D) culture systems. Semin. Cancer Biol. 2005, 15(5), 405–12.
2. Cukierman, E.; Pankov, R.; Yamada, K.M. Cell interactions with three-dimensional matrices. Curr. Opin. Cell Biol. 2002, 14(5), 633–40.
3. Nelson, C.M.; Bissell, M.J. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Ann. Rev. Cell Dev. Biol. 2006, 22, 287–309.
4. Schuetz, E.G.; Li, D.; Omiecinski, C.J.; Muller-Eberhard, U.; Kleinman, H.K.; Elswick, B.; Guzelian, P.S. Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix. J. Cell Physiol. 1988, 134(3), 309–23.
5. Barcellos-Hoff, M.H.; Aggeler, J.; Ram, T.G.; Bissell, M.J. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development 1989, 105(2), 223–35.

6. Webber, M.M.; Bello, D.; Kleinman, H.K.; Hoffman, M.P. Acinar differentiation by non-malignant immortalized human prostatic epithelial cells and its loss by malignant cells. Carcinogenesis 1997, 18(6), 1225–31.

7. Grant, D.S.; Tashiro, K.; Segui-Real, B.; Yamada, Y.; Martin, G.R.; Kleinman, H.K. Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro. Cell 1989, 58(5), 933–43.
8. Griffith, L.G.; Swartz, M.A. Capturing complex 3-D tissue physiology in vitro. Nat. Rev. Mol. Cell Biol. 2006, 7(3), 211–24.
9. Petersen, O.W.; Ronnov-Jessen, L.; Howlett, A.R.; Bissell, M.J. Interaction with Basement Membrane Serves to Rapidly Distinguish Growth and Differentiation Pattern of Normal and Malignant Human Breast Epithelial Cells. Proceedings of the National Academy of Sciences 1992, 89(19), 9064–8.
10. Fridman, R.; Giaccone, G.; Kanemoto, T.; Martin, G.R.; Gazdar, A.F.; Mulshine, J.L. Reconstituted Basement Membrane (Matrigel) and Laminin Can Enhance the Tumorigenicity and the Drug Resistance of Small Cell Lung Cancer Cell Lines. Proceedings of the National Academy of Sciences 1990, 87(17), 6698–6702.
11. Hodkinson, P.S.; Elliott, T.; Wong, W.S.; Rintoul, R.C.; Mackinnon, A.C.; Haslett, C.; Sethi, T. ECM overrides DNA damage-induced cell cycle arrest and apoptosis in small-cell lung cancer cells through beta1 integrin-dependent activation of PI3-kinase. Cell Death Differ. 2006, 13(10), 1776–88.
12. Pogany, G.; Timar, F.; Olah, J.; Harisi, R.; Polony, G.; Paku, S.; Bocsi, J.; Jeney, A.; Laurie, G.W. Role of the basement membrane in tumor cell dormancy and cytotoxic resistance. Oncology 2001, 60(3), 274–81.

Series brought to you by Trevigen, Inc.

What is 3D Culture? - part 2

Three-dimensional cultures are in vitro cell culture models in which extracellular matrix (ECM) proteins recapitulate the physiological cell environment to promote cell differentiation and tissue architecture as seen in vivo. These cultures exhibit considerable similarities in their gene expression profiles; morphology; cell–cell and cell–matrix interactions; and differentiation to their originating tissues, while there are significant differences for cells grown on flat 2-D plastic substrata. (1–3) Under 3D culture conditions, epithelial cells organize to resemble their subsequent tissues, (4–6) endothelial cells arrange to form vascular networks, (7) stromal cells maintain a mesenchymal phenotype, (8) and tumor cells assemble into disorganized masses.(9) In these models, the extracellular environment dictates the cellular program and subsequent differentiation and structure formation; thus it is important to select the right ECM proteins based on cell type and function. Cells of epithelial and endothelial origin have an underlying basement membrane orchestrating their growth and development, and this microenvironment is most commonly reconstructed using a basement membrane extract (BME). Alternatively, mesenchymal cells reside in collagen rich connective tissues, and purified collagen I is most commonly used to mimic these surroundings. Other soluble factors may also be necessary to direct cellular differentiation; these include serum, growth factors, hormones, and other cytokines. Most 3D models have optimized conditions to promote proper differentiation, and these parameters can often be found in the scientific literature. Reagents for research utilizing 3D techniques can be found at www.trevigen.com.

References
1. Birgersdotter, A.; Sandberg, R.; Ernberg, I. Gene expression perturbation in vitro—a growing case for three-dimensional (3-D) culture systems. Semin. Cancer Biol. 2005, 15(5), 405–12.

2. Cukierman, E.; Pankov, R.; Yamada, K.M. Cell interactions with three-dimensional matrices. Curr. Opin. Cell Biol. 2002, 14(5), 633–40.

3. Nelson, C.M.; Bissell, M.J. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Ann. Rev. Cell Dev. Biol. 2006, 22, 287–309.

4. Schuetz, E.G.; Li, D.; Omiecinski, C.J.; Muller-Eberhard, U.; Kleinman, H.K.; Elswick, B.; Guzelian, P.S. Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix. J. Cell Physiol. 1988, 134(3), 309–23.

5. Barcellos-Hoff, M.H.; Aggeler, J.; Ram, T.G.; Bissell, M.J. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development 1989, 105(2), 223–35.

6. Webber, M.M.; Bello, D.; Kleinman, H.K.; Hoffman, M.P. Acinar differentiation by non-malignant immortalized human prostatic epithelial cells and its loss by malignant cells. Carcinogenesis 1997, 18(6), 1225–31.

7. Grant, D.S.; Tashiro, K.; Segui-Real, B.; Yamada, Y.; Martin, G.R.; Kleinman, H.K. Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro. Cell 1989, 58(5), 933–43.

8. Griffith, L.G.; Swartz, M.A. Capturing complex 3-D tissue physiology in vitro. Nat. Rev. Mol. Cell Biol. 2006, 7(3), 211–24.

9. Petersen, O.W.; Ronnov-Jessen, L.; Howlett, A.R.; Bissell, M.J. Interaction with Basement Membrane Serves to Rapidly Distinguish Growth and Differentiation Pattern of Normal and Malignant Human Breast Epithelial Cells. Proceedings of the NationalAcademy of Sciences 1992, 89(19), 9064–8.

3D Culture Series - Part 1

Over the years, cell-based assays have been used by scientists for evaluating factors affecting disease and designing therapies to treat disease states. More recently, we have learned that the artificial cell environment provided by traditional plastic tissue culture impacts the cells’ biological program, and as a result, the information generated by these studies does not readily translate to living organisms. To address this deficiency, 3D culture models have been constructed that mimic the cells’ natural environment, promoting cellular differentiation as seen within living organisms. In these models, cells assemble into organotypic structures exhibiting both structural and functional features of their emanating tissues. While these models provide great advantages regarding efficacy, the presence of cell scaffolding proteins has complicated the adaptation of traditional techniques for analyzing cell culture.

This series will cover many aspects of 3D Culture including:

1. Background of 3D Culture
2. Assessing 3D Culture Cell Proliferation
3. Optimization of 3D Culture Cell Proliferation
4. Determining a Dose Response in 3D Culture

Additional information on 3D Products can be found at www.trevigen.com.

New Citations using Trevigen Products - October 2009

Senescence-Associated Oxidative DNA Damage Promotes the Generation of Neoplastic Cells
Karo Gosselin, Sébastien Martien, Albin Pourtier, Chantal Vercamer, Peter Ostoich, Luc Morat, Laure Sabatier, Laurence Duprez, Claire T'Kint de Roodenbeke, Eric Gilson, Nicolas Malaquin, Nicolas Wernert, Predrag Slijepcevic, Marjan Ashtari, Fazia Chelli, Emeric Deruy, Bernard Vandenbunder, Yvan De Launoit, and Corinne Abbadie
Cancer Res., Oct 2009; 69: 7917 – 7925.
...suspension was immediately laid onto a Trevigen cometslide. Agarose was allowed to solidify...and stained with either SYBR Green I (Trevigen) according to the manufacturers recommendations...oxo-7,8-dihydroguanine (8-oxoG) antibody is from Trevigen. Because it recognizes 8-oxoG in both...


Glyceraldehyde 3-Phosphate Dehydrogenase Depletion Induces Cell Cycle Arrest and Resistance to Antimetabolites in Human Carcinoma Cell Lines
Manali S. Phadke, Natalia F. Krynetskaia, Anurag K. Mishra, and Evgeny Krynetskiy
J. Pharmacol. Exp. Ther., Oct 2009; 331: 77 – 86.
...were detected by Neutral Comet assay with the CometAssay kit (Trevigen, Gaithersburg, MD), according to the manufacturer's instructions...Fragment Length Analysis using Repair Enzymes (FLARE) slides (Trevigen). After cell lysis in a neutral lysis buffer at 4C overnight...


Anti-angiogenic activity of carebastine: a plausible mechanism affecting airway remodelling
A. De Luisi, G. Mangialardi, R. Ria, G. Acuto, D. Ribatti, and A. Vacca
Eur. Respir. J., Oct 2009; 34: 958 – 966.
...24-well plates pre-coated with Cultrex basement membrane extract Trevigen, Inc., Gaithersburg, MD, USA; 400uLwell in 1mLwell of SFM...endothelial cells were seeded on Cultrex basement membrane extract Trevigen, Inc., Gaithersburg, MD, USA in: a serum-free medium SFM alone...


Udu Deficiency Activates DNA Damage Checkpoint
Chiaw-Hwee Lim, Shang-Wei Chong, and Yun-Jin Jiang
Mol. Biol. Cell, Oct 2009; 20: 4183 – 4193.
...assay was performed using the CometAssay kit (4250-040-K; Trevigen, Gaithersburg, MD) according to the manufacturer's instruction. DNA was stained with SYBR Green as provided (Trevigen). Comet images were taken by fluorescence microscopy...


Hepatitis B and Hepatitis C virus replication up-regulate serine protease inhibitor Kazal, resulting in cellular resistance to serine protease dependent apoptosis
Jason Lamontagne, Mark Pinkerton, Timothy M. Block, and Xuanyong Lu
J. Virol., Oct 2009; 10.1128/JVI.01249-09.
...BFA/CHX/Z-VAD122 7 treated cells were washed with PBS and then incubated with annexin V-FITC in 100ul of binding123 buffer (Trevigen Inc., Gaithersburg, MD). After incubating for 30 minutes, the cells were washed124 with PBS and were then visualized with a...


Proteasome-dependent Processing of Topoisomerase I-DNA Adducts into DNA Double Strand Breaks at Arrested Replication Forks
Chao-Po Lin, Yi Ban, Yi Lisa Lyu, and Leroy F. Liu
J. Biol. Chem., Oct 2009; 284: 28084 – 28092.
...streptomycin (100 mug/ml) in a 30 C incubator with 5% CO2. Neutral Comet Assay The comet assays were performed according to the Trevigen CometAssayTM kit protocol with slight modifications. Cells were pretreated with various inhibitors for 30 min, followed by...


In Utero Exposure to Di-(2-Ethylhexyl) Phthalate Decreases Mineralocorticoid Receptor Expression in the Adult Testis
D. B. Martinez-Arguelles, M. Culty, B. R. Zirkin, and V. Papadopoulos
Endocrinology, Oct 2009; 10.1210/en.2009-0847.
...difluoride membrane. The membrane was incubated with antiserum against MR (see above) and then against GAPDH (2275-PC-100; Trevigen, Gaithersburg, MD). Proteins were detected with a secondary antibody coupled to horseradish peroxidase (goat antimouse, 554002...


Effects of Sp1 overexpression on cultured human corneal stromal cells
Xiang Shen, Jeong-Seok Park, Ye Qiu, Joel Sugar, and Beatrice Y J T Yue
Genes Cells, Oct 2009; 14: 1133 - 1139.
...anti-cathepsin B, anti-a1-PI or anti-glyceraldehyde 3-phosphate dehydrogenase (Trevigen, Gaithersburg, MD, USA). Horseradish peroxidase-conjugated goat anti-rabbit IgG was used as the secondary...


Poly(Adenosine 5'-Diphosphate-Ribose) Polymerase Inhibition Counteracts Multiple Manifestations of Experimental Type 1 Diabetic Nephropathy
Viktor R. Drel, Weizheng Xu, Jie Zhang, Ivan A. Pavlov, Hanna Shevalye, Barbara Slusher, and Irina G. Obrosova
Endocrinology, Oct 2009; 10.1210/en.2009-0628.
...Mouse monoclonal anti-poly(ADP-ribose) antibody was purchased from Trevigen, Inc. (Gaithersburg, MD), rabbit poly-clonal antinitrotyrosine (NT) antibody and mouse monoclonal anti-NT antibody...


Development and Characterization of SSR Markers and Their Use to Assess Genetic Relationships among Alfalfa Germplasms
C. He, Z. L. Xia, T. A. Campbell, and G. R. Bauchan
Crop Sci., Oct 2009; 49: 2176 – 2186.
...PCR products were finally extended at 72C for 5 min. DNA fragments were sized using high-resolution 3% TREVIGEL agarose gels (Trevigen, Inc., Gaithersburg, MD) or the ABI PRISM 3100 Genetic Analyzer. Nomenclature for SSR Markers SSR names were prefixed with...


TSLP Induces Chemotactic and Pro-survival Effects in Eosinophils: Implications in Allergic Inflammation
Chun K Wong, Shuiqing Hu, Phyllis F.Y. Cheung, and Christopher WK Lam
Am. J. Respir. Cell Mol. Biol., Oct 2009; 10.1165/rcmb.2009-0168OC.
...Walkersville, MD). Page 8 of 56 9 Apoptosis assay Apoptosis of eosinophils was assessed by the TACS Annexin V-FITC assay (Trevigen, Gaithersburg, MD, USA) using flow cytometry. The population of viable...


Antagonistic roles of four SR proteins in the biosynthesis of alternatively spliced tissue factor transcripts in monocytic cells
Sajiv Chandradas, Gintaras Deikus, Jonathan G. Tardos, and Vladimir Y. Bogdanov
J. Leukoc. Biol., Oct 2009; 10.1189/jlb.0409252.
...lysates, anti-SR protein mAb 16H3. The blot was reprobed with anti-GAPDH polyclonal antibody (Trevigen, Gaithersburg, MD, USA). (B) In silico analysis of TF exon 5 using web resources ESEfinder...


Oral Poly(ADP-Ribose) Polymerase-1 Inhibitor BSI-401 Has Antitumor Activity and Synergizes with Oxaliplatin against Pancreatic Cancer, Preventing Acute Neurotoxicity
Davide Melisi, Valeria Ossovskaya, Cihui Zhu, Roberta Rosa, Jianhua Ling, Patrick M. Dougherty, Barry M. Sherman, James L. Abbruzzese, and Paul J. Chiao
Clin. Cancer Res., Oct 2009; 15: 6367 – 6377.
...activity of PARP in human pancreatic cancer cell lines and MEFs was measured using a universal colorimetric PARP assay kit (Trevigen). Apoptosis The extent of apoptosis was determined by fragmented DNA detection as previously described by Matassov...


Hepatitis C Viral Proteins induce NADPH Oxidase 4 (Nox4) in a TGF-dependent manner: A new contributor of HCV-induced oxidative stress
Howard E. Boudreau, Suzanne U. Emerson, Agnieszka Korzeniowska, Meghan A. Jendrysik, and Thomas L. Leto
J. Virol., Oct 2009; 10.1128/JVI.01059-09.
...antibody, rabbit polyclonal anti-Actin antibody, rabbit polyclonal anti-GAPDH (Trevigen, Gaithersburg, MD, USA), mouse monoclonal anti-V5, goat polyclonal anti-E2...


Acquired Resistance to Combination Treatment with Temozolomide and ABT-888 Is Mediated by Both Base Excision Repair and Homologous Recombination DNA Repair Pathways
Xuesong Liu, Edward K. Han, Mark Anderson, Yan Shi, Dimitri Semizarov, Gang Wang, Thomas McGonigal, Lisa Roberts, Loren Lasko, Joann Palma, Gui-dong Zhu, Thomas Penning, Saul Rosenberg, Vincent L. Giranda, Yan Luo, Joel Leverson, Eric F. Johnson, and Alexander R. Shoemaker
Mol. Cancer Res., Oct 2009; 7: 1686 – 1692.
...Polyclonal anti-PAR antibody was from Trevigen. Western blot analysis was done using enhanced chemiluminescence detection reagents according to the instructions...


Synergistic effects of autologous cell and hepatocyte growth factor gene therapy for neovascularization in a murine model of hindlimb ischemia
Yasutaka Yamamoto, Takashi Matsuura, Genta Narazaki, Miyoko Sugitani, Kohei Tanaka, Akihiro Maeda, Goshi Shiota, Kenzo Sato, Akio Yoshida, and Ichiro Hisatome
Am J Physiol Heart Circ Physiol, Oct 2009; 297: H1329 - H1336.
...by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) using a commercially available kit (TACS/XL, Trevigen, Gaithersburg, MD). Migration assay. A total of 4 105 aMNCs in 0.5 ml DMEM with 0.4% FBS were seeded in the upper chamber...


Translocator Protein Blockade Reduces Prostate Tumor Growth
Arlee Fafalios, Ardavan Akhavan, Anil V. Parwani, Robert R. Bies, Kevin J. McHugh, and Beth R. Pflug
Clin. Cancer Res., Oct 2009; 15: 6177 – 6184.
...Slides were then loaded on a Dako Autostainer. The primary anti-TSPO (working dilution, 1:350) was a polyclonal rabbit antibody (Trevigen). The immunolabeling procedures were done using Dako EnVision-Labeled Polymer-HRP antirabbit and were carried out according...


Vorinostat Inhibits Brain Metastatic Colonization in a Model of Triple-Negative Breast Cancer and Induces DNA Double-Strand Breaks
Diane Palmieri, Paul R. Lockman, Fancy C. Thomas, Emily Hua, Jeanne Herring, Elizabeth Hargrave, Matthew Johnson, Natasha Flores, Yongzhen Qian, Eleazar Vega-Valle, Kunal S. Taskar, Vinay Rudraraju, Rajendar K. Mittapalli, Julie A. Gaasch, Kaci A. Bohn, Helen R. Thorsheim, David J. Liewehr, Sean Davis, John F. Reilly, Robert Walker, Julie L. Bronder, Lionel Feigenbaum, Seth M. Steinberg, Kevin Camphausen, Paul S. Meltzer, Victoria M. Richon, Quentin R. Smith, and Patricia S. Steeg
Clin. Cancer Res., Oct 2009; 15: 6148 – 6157.
...migration assay Boyden chamber motility assays were conducted as described previously (8). Neutral comet assay kit from Trevigen was used according to the recommendations from the manufacturer. DNA was stained with propidium iodide and digital fluorescent...


A real-time fluorescence method for enzymatic characterization of specialized human DNA polymerases
Dorjbal Dorjsuren, David M. Wilson, III, William A. Beard, John P. McDonald, Christopher P. Austin, Roger Woodgate, Samuel H. Wilson, and Anton Simeonov
Nucleic Acids Res., Oct 2009; 37: e128.
...plates following previously described protocols (27). Enzymes Recombinant human DNA pol beta was either purchased from Trevigen (Gaithersburg, MD) or produced via published protocols (28). Equivalent performance was noted for the enzyme obtained...


Chelation of intracellular iron with the antifungal agent ciclopirox olamine induces cell death in leukemia and myeloma cells
Yanina Eberhard, Sean P. McDermott, Xiaoming Wang, Marcela Gronda, Amudha Venugopal, Tabitha E. Wood, Rose Hurren, Alessandro Datti, Robert A. Batey, Jeffrey Wrana, William E. Antholine, John Dick, and Aaron D. Schimmer
Blood, Oct 2009; 114: 3064 - 3073.
...Membranes were probed with polyclonal rabbit anti-human survivin (1 mug/mL), or with mouse anti-human GAPDH (Trevigen). Secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse IgG (1:10 000 [vol/vol]) and...

New Citations using Trevigen Products

The Recombination Protein RAD52 Cooperates with the Excision Repair Protein OGG1 for the Repair of Oxidative Lesions in Mammalian Cells
Nadja C. de Souza-Pinto, Scott Maynard, Kazunari Hashiguchi, Jingping Hu, Meltem Muftuoglu, and Vilhelm A. Bohr
Mol. Cell. Biol., Aug 2009; 29: 4441 - 4454.
...incubated in primary anti-8-oxoG antibody (Trevigen) at a 1:250 dilution in 10 goat serum-PBS...heterochromatic DNA (7, 32, 40; see also the Trevigen web site). For the control, the results...8-oxoG antibody (Fig. 7C) that was used (Trevigen) in two ways. First, treatment of HeLa...

Poly(ADP-ribose) polymerase inhibitor ABT-888 potentiates the cytotoxic activity of temozolomide in leukemia cells: influence of mismatch repair status and O6-methylguanine-DNA methyltransferase activity
Terzah M. Horton, Gaye Jenkins, Debananda Pati, Linna Zhang, M. Eileen Dolan, Albert Ribes-Zamora, Alison A. Bertuch, Susan M. Blaney, Shannon L. Delaney, Madhuri Hegde, and Stacey L. Berg
Mol. Cancer Ther., Aug 2009; 8: 2232 - 2242.
...a commercially available PARP assay (Trevigen). Cell lysates were prepared from 1 107 leukemia cells using 1 PARP buffer (Trevigen) supplemented with 0.4 mmol/L phenylmethylsulfonyl...in triplicate, and recombinant PARP (Trevigen) was used to produce a standard curve...

Poly(ADP-ribosyl)ation directs recruitment and activation of an ATP-dependent chromatin remodeler
Aaron J. Gottschalk, Gyula Timinszky, Stephanie E. Kong, Jingji Jin, Yong Cai, Selene K. Swanson, Michael P. Washburn, Laurence Florens, Andreas G. Ladurner, Joan W. Conaway, and Ronald C. Conaway
PNAS, Aug 2009; 106: 13770 - 13774.
...pmol) Flag-Alc1 (wild-type, E175Q, or D723A) from HEK293/FRT cells or SF9 cells, 115 ng (1 pmol) Parp1 (Trevigen), recombinant Parg (Trevigen), 34 muM nicotine adenine dinucleotide, and 150 ng mono- or oligonucleosomes from HeLa cells (37...

Differences in Immunolocalization of Kim-1, RPA-1, and RPA-2 in Kidneys of Gentamicin-, Cisplatin-, and Valproic Acid-Treated Rats: Potential Role of iNOS and Nitrotyrosine
Jun Zhang, Peter L. Goering, Parvaneh Espandiari, Martin Shaw, Joseph V. Bonventre, Vishal S. Vaidya, Ronald P. Brown, Joe Keenan, Cormac G. Kilty, Nakissa Sadrieh, and Joseph P. Hanig
Toxicol Pathol, Aug 2009; 37: 629 - 643.
...was detected using the TACS in situ apoptotic detection kit (Trevigen, Inc., Gaithersburg, MD). The TUNEL procedures were as follows...ten minutes. In the final step, uncolored soluble chromogen (Trevigen blue label) was enzymatically converted into an insoluble blue-colored...

Dietary zinc restriction and repletion affects DNA integrity in healthy men
Yang Song, Carolyn S Chung, Richard S Bruno, Maret G Traber, Kenneth H Brown, Janet C King, and Emily Ho
Am. J. Clinical Nutrition, Aug 2009; 90: 321 - 328.
...mixed with 200 muL 0.5% low-melting-point agarose and applied onto microscope slides (Trevigen, Gaithersburg, MD). Slides were stored in lysis buffer (Trevigen, Gaithersburg, MD) and shipped to Oregon State University within 3 d. At Oregon State...

1-Adrenergic Receptors Regulate Neurogenesis and Gliogenesis
Manveen K. Gupta, Robert S. Papay, Chris W. D. Jurgens, Robert J. Gaivin, Ting Shi, Van A. Doze, and Dianne M. Perez
Mol. Pharmacol., Aug 2009; 76: 314 - 326.
...detection. Apoptosis Assay. Neurospheres were grown and analyzed for apoptosis by using the FlowTACS Apoptosis Detection Kit (Trevigen, Gaithersburg, MD). Terminal deoxynucleotidyl transferase end-labeling of the free 3-hydroxyl residues in the fragmented DNA...

PP2A-dependent Dephosphorylation of Replication Protein A (RPA) is Required for the Repair of DNA breaks Induced by Replication Stress
Junjie Feng, Timothy Wakeman, Sheila Yong, Xiaohua Wu, Sally Kornbluth, and Xiao-Fan Wang
Mol. Cell. Biol., Aug 2009; 10.1128/MCB.00191-09.
...repair kinetics of HU-induced DNA breaks was evaluated by the alkaline comet2 assay according to the manufacturer's protocol (Trevigen). Briefly, cells were pulse-3 exposed to HU (0.2 mM, 24 hr) and harvested at various recovery time points for single-4 cell...

CD133+ Glioblastoma Stem-like Cells are Radiosensitive with a Defective DNA Damage Response Compared with Established Cell Lines
Amy M. McCord, Muhammad Jamal, Eli S. Williams, Kevin Camphausen, and Philip J. Tofilon
Clin. Cancer Res., Aug 2009; 15: 5145 - 5153.
...the neutral comet assay was done using a commercially available kit according to the recommendations from the manufacturer (Trevigen) with slight modifications. Briefly, monolayer cultures were irradiated (10 Gy), and at specified times, single-cell suspensions...

Nucleophosmin Redistribution following Heat Shock: A Role in Heat-Induced Radiosensitization
Robert P. Vanderwaal, Leonard B. Maggi, Jr., Jason D. Weber, Clayton R. Hunt, and Joseph L. Roti Roti
Cancer Res., Aug 2009; 69: 6454 - 6462.
...on an Olympus fluorescent microscope. Assay for DSBs. The Neutral Comet Assay was performed using the Comet Assay kit from Trevigen following manufacturers instructions. Proteinase K (Sigma), when used, was added to the lysis solution (0.27 U/mL). Lysis occurred...

A real-time fluorescence method for enzymatic characterization of specialized human DNA polymerases
Dorjbal Dorjsuren, David M. Wilson, III, William A. Beard, John P. McDonald, Christopher P. Austin, Roger Woodgate, Samuel H. Wilson, and Anton Simeonov
Nucleic Acids Res., Aug 2009; 10.1093/nar/gkp641.
...plates following previously described protocols (27). Enzymes Recombinant human DNA pol beta was either purchased from Trevigen Inc. (Gaithersburg, MD) or produced via published protocols (28). Equivalent performance was noted for the enzyme obtained...

Modulation of Carcinogen Metabolism by Nitric Oxide-Aspirin 2 Is Associated with Suppression of DNA Damage and DNA Adduct Formation
Christopher J. MacDonald, Robert Y. S. Cheng, David D. Roberts, David A. Wink, and Grace Chao Yeh
J. Biol. Chem., Aug 2009; 284: 22099 - 22107.
...Single cell gel electrophoresis assay (31) with alkaline electrophoresis was performed according to the CometAssay protocol (Trevigen, Gaithersburg, MD), and under protective yellow lighting. In brief, HepG2 cells were treated with either 10 mum BP or 10 nm...

Synergistic effects of autologous cell and hepatocyte growth factor gene therapy for neovascularization in the murine model of hindlimb ischemia.
Yasutaka Yamamoto, Takashi Matsuura, Genta Narazaki, Miyoko Sugitani, Kohei Tanaka, Akihiro Maeda, Goshi Shiota, Kenzo Sato, Akio Yoshida, and Ichiro Hisatome
Am J Physiol Heart Circ Physiol, Aug 2009; 10.1152/ajpheart.00321.2009.
...terminal deoxynucleotide168 transferase-mediated dUTP nick end labeling (TUNEL) using a commercially169 available kit (TACS/XL, Trevigen, Gaithersburg, MD).170 171 Migration assay172 12 A total of 4 ? 105 aMNC in 0.5 mL of DMEM with 0.4% FBS were seeded173 in...

The cytotoxic T lymphocyte protease granzyme A cleaves and inactivates poly(adenosine 5'-diphosphate-ribose) polymerase-1
Pengcheng Zhu, Denis Martinvalet, Dipanjan Chowdhury, Dong Zhang, Ann Schlesinger, and Judy Lieberman
Blood, Aug 2009; 114: 1205 - 1216.
...and goat polyclonal antibodies to C23 (F-18), N-terminal and C-terminal regions of PARP-1, and PARP-2 (Santa Cruz), and PAR (Trevigen, Inc). HRP-conjugated donkey anti-goat IgG (Santa Cruz), HRP-conjugated donkey anti-rabbit and anti-mouse IgG (Amersham) were...

Requirement for Protein Synthesis at Developing Synapses
Joseph Sebeo, Kuangfu Hsiao, Ozlem Bozdagi, Dani Dumitriu, Yongchao Ge, Qiang Zhou, and Deanna L. Benson
J. Neurosci., Aug 2009; 29: 9778 - 9793.
...Sinai School of Medicine, New York, NY), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (rabbit polyclonal; 1:1000; Trevigen). Fluorescence-tagged secondary antibodies were obtained from Vector Laboratories and Jackson ImmunoResearch. For WB, species-appropriate...

Prolonged Cell Cycle Response of HeLa Cells to Low-Level Alkylation Exposure
Allen G. Schroering, Anbarasi Kothandapani, Steve M. Patrick, Saravanan Kaliyaperumal, Vishal P. Sharma, and Kandace J. Williams
Cancer Res., Aug 2009; 69: 6307 - 6314.
...The slides were then placed into neutralizing solution [0.4 mol/L Tris-HCl (pH 7.5)] for 10 min and stained with SYBR Green (Trevigen). Comets were scored using a Nikon epifluorescence microscope at 200 magnification. Fifty cells were analyzed per slide using...

SIRT1 Promotes Cell Survival under Stress by Deacetylation-Dependent Deactivation of Poly(ADP-Ribose) Polymerase 1
Senthilkumar B. Rajamohan, Vinodkumar B. Pillai, Madhu Gupta, Nagalingam R. Sundaresan, Konstantin G. Birukov, Sadhana Samant, Michael O. Hottiger, and Mahesh P. Gupta
Mol. Cell. Biol., Aug 2009; 29: 4116 - 4129.
...autoradiography or Western blotting with the appropriate antibodies. In experiments where the effect of DNA was examined, activated DNA (Trevigen) or genomic DNA (1 ug/sample) was added to the buffer before incubating PARP1 with radiolabeled NAD. Cell death assay...

Single Cell DNA Damage Assessment

FLARE-based Assessment of DNA Damage...
Trevigen’s unique FLARE™ (Fragment Length Analysis using Repair Enzymes) Assays provide the ability to detect DNA damage in single cells using a variety of DNA repair enzymes in conjunction with Trevigen’s CometAssay® single cell gel electrophoresis kit. To assess the type of DNA damage induced by a putative mutagen, drug, or treatment regimen, cells are harvested after treatment and immobilized in a layer of low melting point agarose on the FLARE Slide.

Features

• FLARE Slides allow direct application of low melting point agarose without base layers or pretreatment for three samples
• Hydrophobic barrier allows easy application of cells directly to slide and treatment with enzyme
• Base specific DNA damage detection/enhanced damage characterization

Oxidative Damage Assessment
Fpg
4040-100-FK
8-oxoguanine, DNA containing formamidopyrimidine moieties

Endonuclease III
4045-01K-FK
Thymine Glycol, 5,6-dihydrothymine, urea, 5-hydroxy-6-hydrothymine, 5,6-dihydrouracil, alloxan, 5-hydroxy-6-hydrouracil, uracil glycol, 5-hydroxy-5-methylhydantoin, 5-hydroxycytosine,5-hydroxyuracil,methyltartonylurea, thymine ring saturated or fragmentation product

hOGG1
4130-100-FK
8-oxoguanine, DNA containing formamidopyrimidine moieties

UV Damage Assessment
T4-PDG
4055-100-FK
Cis-syn isomers of cyclobutane pyrimidine dimers

cv-PDG
4065-100-FK
Cis-syn and trans-syn isomers of cyclobutane pyrimidine dimers

UVDE
4100-100-FK
Cyclobutane pyrimidine dimers, (6-4) photoproducts