Luc-Screen System® Protocol (PN T9019D)
Contents
1. gt Applied RS Biosystems Applied Biosystems 35 Wiggins Avenue Bedford MA 01730 800 542 2369 or 781 271 0045 Press 2 Luc Screen System Extended Glow Bioluminescent Reporter Gene Assay System for Detection of Firefly Luciferase P N 11033 T1034 T1035 Contents Page __ oo PREFACE l a INTRODUCTION lU 2 l SYSTEM COMPONENTS llJe legR 2 ll LUCIFERASE DETECTION PROTOCOL 3 R IV PROTOCOL NOTES LllJe llJ_0 _nnsr V APPENDICES Olllllll ann 4 u A Preparation of Controls A S B Use of Luminometers i C Safety 5 Part Number T9019 Revision D Revision Date October 2008 For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES Literature Citation When describing a procedure for p2. Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files Obtaining MSDSs The MSDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day To obtain MSDSs 1 Go to www appliedbiosystems com click Support then select MSDS 2 In the Keyword Search field enter the chemical name product name MSDS part number or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer Chemical waste safety Chemical waste hazards A N CAUTION HAZARDOUS WASTE Refer to Material Safety Data Sheets and local regulations for handling and disposal A N WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems
3. consult the MSDS Handle chemical wastes in a fume hood After emptying a waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Waste disposal If potentially hazardous waste is generated when you operate the instrument you must Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Biological hazard safety General biohazard WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical contai
4. e 5 mL of Buffer 1 and 5 mL of Buffer 2 are required for one entire 96 well microplate 2 Add Buffer 1 50 uL well to cells in 100 uL of culture medium with or without phenol red see Notes 1 3 3 Add Buffer 2 50 uL well Timing between addition of Buffer 1 and Buffer 2 is not critical 4 Incubate for 10 min at room temperature see Note 4 5 Place microplate in a luminometer and measure for 0 1 1 sec well Light signal should decay with a half life of 4 5 hr IV PROTOCOL NOTES 1 The use of white tissue culture treated microplates is recommended for optimal assay sensitivity Clear bottom white plates can be used to allow microscopic examination of cultures White backing sheets may be applied to the plate bottom prior to signal measurement if desired The absolute light signal intensities will be higher approximately two fold since the white backing reflects light toward the PMT detector but relative signal levels should be unaffected Black plates are not recommended since they will cause reduced light yield due to absorbance of the luminescent signal 2 A 2 4 fold decrease in signal intensity is typically observed in culture medium containing phenol red 3 If desired PBS containing 1 mM Mg and 1 mM Ca can be substituted for culture medium prior to assay 4 For steady light emission the temperature during assay and measurement should be maintained at 23 27 C V APPENDICES A Preparation of Controls Po
5. instruments are potentially hazardous and can cause injury illness or death AN WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical waste safety guidelines To minimize the hazards of chemical waste Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines
6. n that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations MSDSs The MSDSs for any chemicals supplied by Applied Biosystems are available to you free 24 hours a day For instructions on obtaining MSDSs see Obtaining MSDSs on page 6 IMPORTANT For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer How to Obtain Support For the latest services and support information for all locations go to www appliedbiosystems com At the Applied Biosystems web site you can e Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support e Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents e Download PDF documents e Obtain information about customer training e Download software updates and patches I INTRODUCTION Firefly luciferase is widely used as a reporter gene for studying gene regulation gene function and for high throughput compound screening with gene expression assay Luciferase is a very sensitive genetic reporter due to the lack of any endogenous activity in mammalian cells or tissues 1 2 Tropix Luc Screen luciferase system is designed to provide a convenient and easy to use firefly luciferase detection a
7. nment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories stock no 017 040 00547 4 bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx 01 29cfr1910a_ 01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials IMPORTANT Additional information about biohazard guidelines is available at www cdc gov Chemical alerts For the definitions of the alert words IMPORTANT CAUTION WARNING and DANGER see Safety alert words on page 1 General alerts for all chemicals EXAMPLE Avoid contact with skin eyes and or clothing Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Specific chemical alerts N WARNING CHEMICAL HAZARD Buffer 1 may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves WARNING CHEMICAL HAZARD Buffer 2 may cause eye skin and respiratory tract irrita
8. oplates The linear range of detection will vary according to cell type and on the reporter enzyme expression level The number of cells used per well should be optimized to prevent a measurement signal that is outside the linear range of the luminometer Extremely high light signals can saturate the detector very unlikely for experimental samples resulting in erroneous measurements Refer to your luminometer user s manual to determine the upper limit for your specific luminometer Contact Applied Biosystems Technical Support group for additional questions C Safety General chemical safety Chemical hazard warning AN AN AN AN WARNING CHEMICAL HAZARD Before handling any chemicals refer to the Material safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions WARNING CHEMICAL HAZARD All chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument WARNING CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bo
9. s not supplied should be used Applications The Luc Screen reporter gene assay system is ideal for high throughput firefly luciferase reporter assays in transiently and stably transfected mammalian cells for gene expression assays 3 4 compound screening 5 as a read out for cAMP induction in signaling pathways 6 7 and large scale promoter function assays 8 as well as in insect cell lines 4 In addition it has also been used as a read out for viral replication 5 and a viral mediated membrane fusion assay 9 ll SYSTEM COMPONENTS Shelf life for all Luc Screen kit components is 1 yr when stored as indicated Ce T1035 T1033 T1034 Microplate assays 1000 10 000 Buffer 1 2 x 25 mL 2 x 250 mL Buffer 2 2 x 25 mL 2 x 250 mL 1 Buffer 1 Contains buffered luciferase reaction and cell lysis reagents Store at 20 C Buffer can be refrozen or stored at 4 C for one week 2 Buffer 2 Contains luciferin substrate Store at 20 C Buffer can be refrozen or stored at 4 C for one week II LUCIFERASE DETECTION PROTOCOL Please read the entire Protocol and Notes sections before proceeding All assays should be performed in duplicate or triplicate See Appendix A for preparation of positive and negative controls For the following hazards see the complete safety alert descriptions in Appendix C Safety on page 5 A N WARNING CHEMICAL HAZARDS Buffer 1 Buffer 2 1 Allow Buffers 1 and 2 to warm to room temperatur
10. sitive Control Prepare stock enzyme by reconstituting lyophilized luciferase Sigma L 9506 to 1 mg mL in 0 1 M sodium phosphate pH 7 0 0 1 BSA Store aliquots of stock enzyme at 80 C and use without repeated freeze thaw Generate a standard curve by serially diluting the stock enzyme in 100 uL of culture medium or buffer PBS containing 1mM Ca and 1mM Mg used for experimental samples For the high end of the dilution curve use 1 10 ng of enzyme Purified enzyme provides a positive control for the assay reagents as well as a means to determine the range of detection of the luminometer instrumentation if desired The purified enzyme standard curve is not intended or accurate for absolute quantitation of reporter enzyme concentrations as the specific activity of the purified enzyme preparation and the reporter enzyme may differ significantly Additional positive conrols can include use of luciferase constructs that provide constitutive expression of reporter enzyme as a positive control for cell transfection Negative Control Assay an equivalent culture of mock transfected cells to determine assay background In experiments involving induction of reporter expression uninduced cells should be assayed as a negative control for total assay background B Use of Luminometers We recommend using a single mode luminometer or a multi mode detection instrument set for luminescence measurement to measure light emission from 96 or 384 well micr
11. ssay performed directly in microplate cell cultures in the presence of cell culture medium particularly for use with high throughput screening applications Luc Screen conveniently couples in well cell lysis in the presence of culture medium with a high sensitivity assay that exhibits extended glow light emission kinetics Light signal can be measured between 10 minutes and several hours after adding assay reagents Luc Screen system is designed for maximum assay flexibility in a high throughput format and can be used in luminometers without automatic injectors The system provides glow light emission kinetics with a half life of 4 5 hours providing flexibility in the time between reagent addition and measurement Detection of as little as 30 50 fg of luciferase is achieved in the presence of culture medium The assay has a broad dynamic detection range of 50 fg to 100 ng of purified enzyme greater than six orders of magnitude Two reagents are added to cells in culture medium with or without phenol red in microplate wells Although the presence of phenol red causes some decrease in signal intensity assay sensitivity is not affected If desired culture medium can be removed and replaced with PBS containing Ca and Mg Cell lysis occurs during an initial 10 minute incubation within this time light signal reaches plateau The light signal is then measured in a microplate luminometer Solid white or white side clear bottom tissue culture microplate
12. tial expression of the thyrostimulin subunits glycoprotein 2 and 5 in the rat pituitary J Mol Endocrinol 37 39 50 Coleman SL PR Buckland B Hoogendoorn C Guy K Smith and MC O Donovan 2002 Experimental analysis of the annotation of promoters in the public database Human Mol Gen 11 16 1817 1821 Baba M K Takashima H Miyake N Kanzaki K Teshima X Wang M Shiraishi and Y lizawa 2005 TAK 652 inhibits CCR5 mediated Human Immunodeficiency Virus type 1 infection in vitro and has favorable pharmacokinetics in humans Antimicrob Agents Chemother 49 11 4584 4591 For complete updated reference list AB 120MI10 please see http www appliedbiosystems com Product amp Service Literature
13. tion Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves VI REFERENCES Gould SJ and S Subramani 1988 Firefly luciferase as a tool in molecular and cell biology Anal Biochem 175 5 13 Brasier AR JE Tate and JF Habener 1989 Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines Bio Techniques 7 1116 1122 Ahmed CMI KN Wills BJ Sugarman DE Johnson M Ramachandra TL Nagabhushan and JA Howe 2001 Selective expression of nonsecreted interferon by an adenoviral vector confers antiproliferative and antiviral properties and causes reduction of tumor growth in nude mice J Interferon Cytokine Res 21 399 408 Li J and C Y Wang 2008 TBL1 TBLR1 and catenin recruit each other to Wnt taget gen promoter for transcirption activation and oncogenesis Nature Cell Biol 10 2 160 169 Wang X H Miyake M Okamoto M Saito J l Fujisawa Y Tanaka S Izumo and M Baba 2002 Inhibition of the Tax dependent Human T Lymphotropic Virus Type replication in persistently infected cells by the fluoroquinolone derivative K 37 Mol Pharmacol 61 6 1359 1365 Reinscheid RK Y L Xu N Okamura J Zeng S Chung R Pai Z Wang and O Civelli 2005 Pharmacological characgerization of human and murine neuropeptide S receptor variants J Pharmacol Exp Therapeut 315 3 1338 1345 Nagasaki H Z Wang VR Jackson S Lin H P Nothacker and O Civelli 2006 Differen
14. ttles WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical safety guidelines To minimize the hazards of chemicals Read and understand the Material Safety Data Sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page 6 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal MSDSs About MSDSs Chemical manufacturers supply current Material Safety
15. ublication using this product please refer to it as the Luc Screen System Trademarks Applied Biosystems AB Design Luc Screen and Tropix are registered trademarks of Applied Biosystems or its subsidiaries in the US and certain other countries All other trademarks are the sole property of their respective owners Copyright 2008 Applied Biosystems All Rights Reserved PREFACE Safety Information Note For general safety information see this Preface and Appendix C Safety on page 5 When a hazard symbol and hazard type appear by a chemical name or instrument hazard see the Safety Appendix for the complete alert on the chemical or instrument Safety Alert Words Four safety alert words appear in Applied Biosystems user documentation at point in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situatio
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