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Manual: Pathdetect Reporter Cell Lines

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Manual: Pathdetect Reporter Cell Lines PathDetect Reporter Cell Lines INSTRUCTION MANUAL Catalog #800050 (PathDetect HLR Cell Line) #800055 (HLR-Elk1 Cell Line) #800060 (HLR-CHOP Cell Line) #800065 (HLR-CREB Cell Line) Revision A BN #800050-12 For In Vitro Use Only 800050-12 LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this product. No other warranties of any kind, express or implied, including without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by Agilent. Agilent shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product. ORDERING INFORMATION AND TECHNICAL SERVICES United States and Canada Agilent Technologies Stratagene Products Division 11011 North Torrey Pines Road La Jolla, CA 92037 Telephone (858) 373-6300 Order Toll Free (800) 424-5444 Technical Services (800) 894-1304 Internet [email protected] World Wide Web www.stratagene.com Europe Location Telephone Fax Technical Services Austria 0800 292 499 0800 292 496 0800 292 498 Belgium 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 15775 0800 15740 0800 15720 France 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 919 288 0800 919 287 0800 919 289 Germany 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 182 8232 0800 182 8231 0800 182 8234 Netherlands 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 023 0446 +31 (0)20 312 5700 0800 023 0448 Switzerland 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 563 080 0800 563 082 0800 563 081 United Kingdom 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 917 3282 0800 917 3283 0800 917 3281 All Other Countries Please contact your local distributor. A complete list of distributors is available at www.stratagene.com. PathDetect Reporter Cell Lines CONTENTS Materials Provided.............................................................................................................................. 1 Storage Conditions.............................................................................................................................. 1 Additional Materials Required .......................................................................................................... 2 Safety Consideration........................................................................................................................... 2 Notices to Purchaser ........................................................................................................................... 2 Introduction......................................................................................................................................... 3 Preprotocol Considerations................................................................................................................ 5 Choosing a Transfection Method .......................................................................................... 5 Tissue Cultureware................................................................................................................ 5 Designing the Experiment ..................................................................................................... 5 Cell Culture Protocols......................................................................................................................... 7 Starting the Cells in Culture .................................................................................................. 7 Passaging the Cells................................................................................................................ 7 Freezing the Cells for Long-term Storage ............................................................................. 8 Preparing the Cells for Transfection................................................................................................. 9 Preparing Plasmids for Transfection ................................................................................................ 9 Studying the Effects of a Gene Product................................................................................. 9 Transfecting the Cells ......................................................................................................................... 9 Preparing Cell Lysates for Luciferase Activity Assay ................................................................... 10 Performing the Luciferase Activity Assay ...................................................................................... 10 Troubleshooting ................................................................................................................................ 11 Appendix I: Plasmid Information.................................................................................................... 12 The pFA trans-Activator Plasmids...................................................................................... 12 Preparation of Media and Reagents................................................................................................ 13 References.......................................................................................................................................... 14 Endnotes............................................................................................................................................. 14 MSDS Information............................................................................................................................ 14 Quick-Reference Protocol ................................................................................................................ 15 PathDetect Reporter Cell Lines MATERIALS PROVIDED Catalog Component Concentration Quantity #800050 HLR Cell Line — 1 × 106 viable cells pFA2-CREB fusion trans-activator plasmid 25 ng/μl 5 μg pFC-PKA plasmid (positive control) 25 ng/μl 5 μg pFC-CMV negative control plasmid 25 ng/μl 5 μg Catalog Component Concentration Quantity #800055 HLR-Elk1 trans-Reporting Cell Line — 1 × 106 viable cells pFC-CMV negative control plasmid 25 ng/μl 5 μg pFC-MEK1 plasmid (positive control) 25 ng/μl 5 μg Catalog Component Concentration Quantity #800060 HLR-CHOP trans-Reporting Cell Line — 1 × 106 viable cells pFC-CMV negative control plasmid 25 ng/μl 5 μg pFC-MEK3 plasmid (positive control) 25 ng/μl 5 μg Catalog Component Concentration Quantity #800065 HLR-CREB trans-Reporting Cell Line — 1 × 106 viable cells pFC-CMV negative control plasmid 25 ng/μl 5 μg pFC-PKA plasmid (positive control) 25 ng/μl 5 μg STORAGE CONDITIONS Control Plasmids: –20°C Cell lines: Liquid Nitrogen Revision A © Agilent Technologies, Inc. 2008. PathDetect Reporter Cell Lines 1 ADDITIONAL MATERIALS REQUIRED Growth medium (See Preparation of Media and Reagents) Luciferase assay kit 5-ml BD Falcon polystyrene round-bottom tubes (BD Biosciences Catalog #352054) Calcium- and magnesium-free PBS Tissue culture plasticware Transfection reagents Luminometer Hygromycin G418 Sulfate SAFETY CONSIDERATION The American Type Culture Collection (ATCC) has designated CCL-2 HeLa cell lines as Biosafety Level 2. Appropriate caution should be used while handling any human-derived cell line. For more information, see Biosafety in Microbiological and Biomedical Laboratories (4th edition). It is available on the Web sites of both the National Institutes of Health at http://bmbl.od.nih.gov and the Office of Health and Safety of the Centers for Disease Control and Prevention at www.cdc.gov/od/ohs (Click the link to Biosafety Information). NOTICES TO PURCHASER The use of the CMV Promoter is covered under U.S. Patent Nos. 5,168,062 and 5,385,839 owned by the University of Iowa Research Foundation and licensed FOR RESEARCH USE ONLY. For further information, please contact UIRF at 319-335-4546. A license (from Promega for research reagent products and from The Regents of the University of California for all other fields) is needed for any commercial sale of nucleic acid contained within or derived from this product. The HLR, HLR-cJun, HLR-Elk1, HLR-CHOP, and HLR-CREB cell lines are proprietary to Agilent. Purchase of these cell lines includes a limited non-exclusive license under Agilent’s proprietary rights to use these cell lines for research purposes only. This license does not grant any rights to: (i) modify any of these cell lines; (ii) offer any of these cell lines, or any derivatives or modifications thereof, for resale; or (iii) otherwise distribute or transfer any of these cell lines, or any derivative or modification thereof, to third parties for any purpose or use. No other license, express, implied or by estoppel, is granted. 2 PathDetect Reporter Cell Lines INTRODUCTION The Stratagene PathDetect reporter cell lines are designed for specific, rapid, and convenient analysis of the in vivo activation of transcriptional activators and upstream signal transduction pathways.1,2 Two types of stable reporter cell lines have been developed for use with the PathDetect system using ATCC, CCL-2 HeLa cells. The first type is the HeLa Luciferase Reporter (HLR) cell line, which contains a stably integrated luciferase reporter gene. The second type includes three HLR trans-reporting cell lines, which contain both the luciferase reporter gene and the gene for a trans-acting GAL4 fusion protein (Elk1, CHOP, or CREB). The cell lines are useful for examining whether and where a newly cloned gene is involved in a signal transduction pathway or to study the effects of growth factors,
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