C ELL CANCER CELL ASSAYS. Cell Adhesion. Cell Invasion/ Cell Migration. Endothelial Cell Invasion. Tube Formation. 3-D Culture.

Transcription

1 C ELL I N VA S I O N P RO D U C T S Cell Adhesion Cell Invasion/ Cell Migration Endothelial Cell Invasion Tube Formation Angiogenesis CANCER CELL ASSAYS 3-D Culture

2 CA N C ER CELL AS S AYS The behavior of cancer cells has both intrigued and plagued scientists for years. As a provider of tools for cancer research, Trevigen has developed and optimized extracellular matrix based assay formats to assess the behavior of cancer cells. Keeping the researcher in mind, we emphasized sensitivity, accuracy, and ease of use. The culmination of this development work is a series of products and methods designed to study cancer progression at the cellular level. These include assays that measure the critical cellular functions of adhesion, proliferation, migration, and invasion, as well as 3-D assays that may be used to assess cellular differentiation, morphology, angiogenic potential, and molecular composition of cells within their physiological microenvironment. CELL INVASION Trevigen s Cell Invasion Assays quantitate the degree to which invasive cells penetrate a physiological barrier. Assay Procedure Cultrex and CultreCoat Cell Invasion Assays accelerate the screening process for compounds that influence cell invasion through in vitro extracellular matrices. These assays offer a flexible, standardized, high- throughput format for quantitating the degree to which invasive cells penetrate a barrier consisting of extracellular matrix proteins in response to chemoattractants and/or inhibiting compounds. The assay has been functionally tested with MCF-7, HT-1080, NIH-3T3, and MDA-MB-231 cell lines. Cells BME Coating Media with or without chemoattractants Cells invading barrier Fluorescence read directly from plates Cells dissociated & fluorescence labeled 50% Size VWR Cat. No. CultreCoat BME Cell Invasion Assay Cultrex BME Cell Invasion Assay Cultrex Collagen I Cell Invasion Assay Cultrex Collagen IV Cell Invasion Assay Cultrex Laminin I Cell Invasion Assay Cultrex BME Cell Invasion Assay Cultrex Collagen I Cell Invasion Assay Cultrex Collagen IV Cell Invasion Assay Cultrex Laminin I Cell Invasion Assay Invasion (%) 40% Description 30% 20% 10% 0% DMEM 10% FBS MCF-7 1 DMEM 10% FBS HT-1080 Quantitation of the ability of the human fibroblastic cell line, HT-1080, to cross a barrier consisting of an 8 micron polycarbonate filter occluded with Cultrex Basement Membrane Extract over a 24 hour period in response to 10% FBS. Samples were run in triplicate for both HT-1080 and the non-invasive MCF-7, mammary epithelial cell line.

3 CELL MIGRATION Trevigen s Cell Migration Assays quantitate the degree to which cells migrate in response to chemoattractants and/or inhibiting stimulant compounds. Trevigen s Cultrex Cell Migration Assay utilizes a simplified Boyden chamber design with an 8 micron polyethylene terephthalate (PET) membrane. Ports within the migration chamber (top) allow access to the assay chamber (bottom). The inserts are sufficiently large to allow for subsequent analysis of migrating subpopulations, in response to chemokines, cytokines, drugs or other compounds of interest. Description Size VWR Cat. No. Cultrex Cell Migration Assay Cultrex Cell Migration Assay CELL ADHESION Trevigen s CultreCoat Cell Adhesion Assays are a simple, standardized, high-throughput format for assessing factors that influence cell-matrix interactions. The extracellular matrix (ECM) provides a structural and signaling framework for tissue morphogenesis, homeostasis, and wound healing by regulating cellular activities. These activities are modulated through interactions between specific transmembrane cell surface receptors, which are most commonly integrins, and their coordinate ECM ligands, resulting in cellular adhesion and signal transduction. During this process, the extracellular domain of the integrin binds to the ECM, and the intracellular domain binds to the cytoskeleton, forming a bridge across the plasma membrane. Concurrently, this interaction controls a wide variety of cellular processes, including cell cycle progression, differentiation, cell migration and invasion, and apoptosis. Background Control (No Cells) Samples Specificity Control (No ECM Coating) Kit Features The black stripwell format minimizes background, providing greater sensitivity, and it affords flexibility for the number of samples assessed. Multiple experiments can be conducted simultaneously using the same kit. Calcein labeling allows direct comparisons between the number of cells that are loaded and the number that adhere, providing a loading control. Controls are provided for determining background and non-specific binding. The CultreCoat Cell Adhesion Assays have been adapted to multiple formats so that cell adhesion may be evaluated against different extracellular matrices; the assay is available in seven formats. The Adhesion Protein Array Kit allows the researcher to assess adhesion on BME, Laminin, Collagen I, Collagen IV, Fibronectin, and Vitronectin at the same time. Adhesion (%) Description Size VWR Cat. No. CultreCoat Adhesion Protein Array Kit 96 Wells CultreCoat BME Cell Adhesion Assay 96 Wells CultreCoat Collagen I Cell Adhesion Assay 96 Wells CultreCoat Collagen IV Cell Adhesion Assay 96 Wells CultreCoat Fibronectin Cell Adhesion Assay 96 Wells CultreCoat Laminin I Cell Adhesion Assay 96 Wells CultreCoat Vitronectin Cell Adhesion Assay 96 Wells % 80% 70% 60% 50% 40% 30% 20% 10% 0% Collagen IV MG63 Laminin I BME Collagen I HT-1080 Fibronectin Vitronectin Percent adhesion for MG63, human osteosarcoma, to Collagen IV, Laminin I, and BME, and HT1080, human fibrosarcoma, to Collagen I, Fibronectin, and Vibronectin. Samples were assessed in duplicate using 15,000 cells/well for an adhesion period of 1 hour and 15 minutes. 2

4 ENDOTHELIAL CELL INVASION Trevigen s Cultrex Endothelial Cell Invasion Assay accelerates the screening process for compounds that influence vascular endothelial cell invasion through the basement membrane. The Endothelial Cell Invasion kit offers a flexible, standardized, high-throughput format for quantitating the degree to which endothelial cells penetrate an in vitro BME barrier in response to chemoattractants and/or inhibiting compounds. This assay employs a simplified Boyden chamber-like design with an 8 micron polyethylene terephthalate (PET) membrane. Sulforaphane, a naturally occurring cancer chemopreventive agent, is provided as a control for inhibition of in vitro endothelial cell migration/invasion on Cultrex BME. Ports within the migration/invasion chamber (top) allow access to the assay chamber (bottom) without dismantling the device. This design is easier to use, prevents contamination, and is adaptable for robotic high-throughput systems. The assay chamber may be directly analyzed in a 96 well plate reader, eliminating transfer steps that introduce additional variablity to the assay. The permissiveness of the BME matrix may also be optimized to fit each experiment by adjusting the coating concentration. Cell Invasion Plate Exploded View Chamber Cover % of migration control Description Size VWR Cat. No. Cultrex In Vitro Angiogenesis Assay Endothelial Cell Invasion Kit 96 Tests EBM EGM-2 EGM-2 + 5µM Sulforaphane SVEC4-10 HUVEC HBMVEC Relative quantitation (as a percent of the migration control) of the ability of endothelial cell lines (SVEC4-10, HUVEC and HBMVEC) to cross an 8 micron PET membrane coated with 1X BME solution over a 24 hour period in response to VEGF, bfgf and other chemoattractants contained in the EGM-2 medium, in the presence or absence of 5 µm Sulforaphane. Receiver Plate CITATIONS Pancreatic Stellate Cells: Partners in Crime with Pancreatic Cancer Cells Cell Invasion Alain Vonlaufen, Swapna Joshi, Changfa Qu, Phoebe A. Phillips, Zhihong Xu, Nicole R. Parker, Cheryl S. Toi, Romano C. Pirola, Jeremy S. Wilson, David Goldstein, and Minoti V. Apte Cancer Res., Apr 2008; 68(7): Requirement of HMGB1 for stromal cell-derived factor-1/cxcl12 -dependent migration of macrophages and dendritic cells Lara Campana, Lidia Bosurgi, Marco E. Bianchi, Angelo A. Manfredi, and Patrizia Rovere-Querini J. Leukoc. Biol., May 2009; 86(3): Cell Migration A soluble form of the receptor for advanced glycation endproducts (RAGE) is produced by proteolytic cleavage of the membrane-bound form by the sheddase a disintegrin and metalloprotease 10 (ADAM10) Cell Migration Angela Raucci, Simona Cugusi, Antonella Antonelli, Silvia M. Barabino, Lucilla Monti, Angelika Bierhaus, Karina Reiss, Paul Saftig, and Marco E. Bianchi FASEB J, Oct 2008; 22(10):

5 FAQS 1. What is cell migration? Cell migration is the movement of cells in response to a chemical stimulus; this is also known as chemotaxis. Cultrex Cell Migration Assays evaluate cell migration based on the cells ability to traverse an uncoated membrane with 8 micron pores, in response to a chemotactic gradient. The cells must undergo cyto-skeletal remodeling to fit into the pores and pull themselves through to the underside of the membrane. 2. What is cell invasion? Cell invasion is cell migration through a physiological barrier in response to a chemoattractant, and this recapitulates cell movement within a physiological environment which is composed of extracellular matrix proteins. Here, the membranes are coated with a layer of extracellular matrix proteins, and the cells must traverse this barrier through a combination of protein degradation and cellular locomotion. 3. Can transwells be re-used and can wells be used at different times? For optimum performance, the transwells should only be used once. For the 96 well format, sterile sealing tape may be used to reserve wells for later use. 4. Which matrix should I use for cell invasion? Choice of matrix should correspond to the environment that you wish to recapitulate. Basement membrane extract (BME) will recapitulate the basal lamina, which underlie most cells of epithelial or endothelial origin. Collagen I is the major constituent of connective tissue, and it is commonly inhabited by stationary cells, such as fibrocytes and adipose cells, as well as migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils. 5. What are the variables associated with cell invasion? The major variables associated with cell invasion are cell type, cell density, composition of the physiological barrier, thickness of the physiological barrier, chemoattractant that is used, and time of culture. 6. Should the wells used for the standard curve be assayed at the same time as the rest of the plate? Yes. After incubating the test wells for hours, the wells designated for the standard curve should be treated and incubated for one hour. 7. Can the migration kit be used for T cells/leukocytes? The protocol and directions provided with the kit are not written to support assaying with nonadherent cells, such as resting T and B leukocytes. Optimization would be required for induction of adherence, and addition of Calcein-AM at the beginning of the assay may be required instead of at the end. 8. Do we need to scrape/scrub off cells from the top of the transwells? The cells in the top chamber are too large to transcend the pores in the membrane without cytoskeletal rearrangement, and there is no chemotactic gradient present during Calcein-AM labeling/detachment for detection, hence making scraping cells off the top of the transwells unnecessary. For more FAQs, please visit /faqs/faq_sections.php TECHNICAL PRESENTATIONS Trevigen has numerous technical references available at including scientific posters, technical presentations, and scientific papers. 4

6 WHAT CUSTOMERS SAY Vivian Barry, Scientist Arresto Biosciences My company has been using Trevigen s Cultrex Collagen I and IV Cell Invasion Assays to screen numerous compounds. We have found that these kits are reliable and the results are always consistent. The dependability of these kits is essential when screening a variety of compounds in a limited amount of time. We had tested a variety of cell invasion assay kits on the market and found that nothing compared to the quality of Trevigen s Cell Invasion Assays. Lan Chun Tu, PhD Research Scientist Institute for Systems Biology The CultreCoat plates worked out great. I have struggled with the cell invasion assay for a long time. With the CultreCoat Cell Invasion Assay from your company, I finally can close my project. The assay is nice and easy and most importantly, the variation between replicates is small and bias is minimal. RELATED PRODUCTS Natural Proteins VWR Cat. No. Cultrex Bovine Collagen I Cultrex Rat Collagen I Cultrex Mouse Collagen IV Cultrex Mouse Laminin I Cultrex Mouse Laminin I, PathClear Cultrex Human Fibronectin, PathClear Cultrex Bovine Fibronectin Cultrex Human Vitronectin, PathClear Cultrex Bovine Vitronectin Amino Acids VWR Cat. No. Cultrex Poly-L-Lysine Cultrex Poly-D-Lysine Basement Membrane Extract VWR Cat. No. Cultrex BME with Phenol Red Cultrex BME without Phenol Red Cultrex BME Reduced Growth Factor with Phenol Red Cultrex BME Reduced Growth Factor without Phenol Red Cultrex Human PathClear BME without Phenol Red Cultrex PathClear BME with Phenol Red Cultrex PathClear BME without Phenol Red Cultrex PathClear Reduced Growth Factor BME with Phenol Red Cultrex PathClear Reduced Growth Factor BME without Phenol Red Cultrex PathClear HC20+ BME High Protein Concentration BME Cultrex 3D Culture Matrix BME Trevigen, Inc. is a rapidly growing biotechnology company focused on the development of products and technology for cancer research, emphasizing apoptosis, DNA damage and repair, and cell function and behavior. Trevigen has been a long-standing provider of quality reagents and kits for researchers investigating programmed cell death and DNA damage and repair. A logical extension of our focus on cancer research has been the recent development of assays for cell function and behavior including angiogenesis, cell invasion and tumor formation. Currently, our product portfolio contains over 500 products categorized into four processes Apoptosis, DNA Damage and Repair, Cancer Cell Behavior, and Oxidative Stress Trevigen, Inc. Trevigen, Cultrex, CultreCoat and PathClear are registered trademarks and HC20+ and Culture Matrix are trademarks of Trevigen, Inc. All images property of Trevigen, Inc. unless noted. 0610