Phospha-Light™ System Protocol (PN T9007D)
Contents
1. Applied KS Biosystems Applied Biosystems 35 Wiggins Avenue Bedford MA 01730 800 542 2369 781 271 0045 Press 2 http www appliedbiosystems com Phospha Light System Chemiluminescent Reporter Gene Assay System for Detection of Placental Alkaline Phosphatase P N T1015 T1016 T1017 Contents Page PREFACE l INTRODUCTION II SYSTEM COMPONENTS Ill DETECTION PROTOCOL A Detection with Tube Luminometers B Detection with Microplate Luminometers C Extract Preparation for Non Secreted Placental Alkaline Phosphatase D Direct Lysis Procedure for Microplate Cultures E Protocol Notes IV APPENDICES A Preparation of Controls B Use of Luminometers C Safety REFERENCES ONNNDOONTAARHRWD N Part Number T9007 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 FORES2. 18 19 tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves REFERENCES Berger J J Hauber R Hauber R Geiger and BR Cullen 1988 Secreted placental alkaline phosphatase A powerful new quantitative indicator of gene expression in eukaryotic cells Gene 66 1 10 Cullen B and M Malim 1992 Secreted placental alkaline phosphatase as a eukaryotic reporter gene Meth Enzymol 216 362 368 Bronstein JJ Fortin JC Voyta RR Juo B Edwards CEM Olesen N Lijam and LJ Kricka 1994 Chemiluminescent reporter gene assays Sensitive detection of the GUS and SEAP gene products BioTechniques 17 1 172 178 Bronstein CS Martin JJ Fortin CEM Olesen and JC Voyta 1996 Chemiluminescence sensitive detection technology for reporter gene assays Clin Chem 42 9 1542 1546 Treeck O G Pfeiler D Mitter C Lattrich G Piendl and O Ortmann 2007 Estrogen receptor B1 exerts antitumoral effects on SK OV 3 ovarian cancer cells J Endocrin 193 421 433 Feehan C K Darlak J Kahn B Walchek AF Spatola and TK Kishimoto 1996 Shedding of the lymphocyte L selectin adhesion molecule is inhibited by a hydroxamic acid based protease inhibitor J Biol Chem 271 12 7019 7024 Andr s V S Fisher P Wearsch and K Walsh 1995 Regulation of Gax homeobox gene transcription by a combination of positive factors including myocyte specific enhancer factor 2 Mol Cell Biol 15 8
3. 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 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 applicati
4. 4272 4281 Guo K and K Walsh 1997 Inhibition of myogenesis by multiple cyclin cdk complexes J Biol Chem 272 2 791 797 Benham FJ J Fogh and H Harris 1981 Alkaline phosphatase expression in human cell lines derived from various malignancies Int J Cancer 27 5 637 644 Brown MA Q Zhao KA Baker C Naik C Chen L Pukac M Singh T Tsareva Y Parice A Mahoney V Roschke Sanyal and S Choe 2005 Crystal structure of BMP 9 and functional interactions with pro region and receptors J Biol Chem 280 26 25111 25118 Zhang J J Ou Y Bashmakov JD Horton MS Brown and JL Goldstein 2001 Insulin inhibits transcription of IRS 2 gene in rat liver through an insulin response element IRE that resembles IREs of other insulin repressed genes Proc Natl Acad Sci USA 98 7 3756 3761 Poser S S Impey Z Xia and DR Storm 2003 Brain derived neurotrophic factor protection of cortical neurons from serum withdrawal induced apoptosis is inhibited by cAMP J Neurosci 23 11 4420 4427 Cao H A Wang B Martin DR Koehler PL Zeitlin AKTanawell and J Hu 2005 Down regulation of IL 8 expression in human airway epithelial cells through helper dependent adenoviral mediated RNA interference Cell Research 15 2 111 119 Hobbs WE DE Brough Kovesdi and NA DeLuca 2001 Efficient activation of viral genomes by levels of Herpes Simplex Virus ICPO insufficient to affect cellular gene expression or cell survival J Virol 75 7 3391 3403 Y
5. or viral vector infected animals The Phospha Light reporter gene assay system has been used to measure SEAP levels in mouse 21 rat 22 marmoset 23 monkey 24 and pig sera 25 and in chicken egg allantoic fluid 26 The mouse SEAP protein mSEAP has recently been developed for improved SEAP protein stability in transgenic mice and Phospha Light system has been used for sensitive detection of MSEAP 21 Beyond reporter gene gene expression applications the Phospha Light assay system is used to measure SEAP as a functional reporter for receptor ligand binding assays with a SEAP ligand chimera 27 protease mediated secretion 28 and for secretion pathway activity 29 30 including as a functional assay to measure effects of siIRNA medated protein knockdown on specific protein secretion pathways 31 Finally it has been used for the cellular measurement of non placental alkaline phosphatase as a biomarker 10 Il SYSTEM COMPONENTS Shelf life for all Phospha Light kit components is 1 yr at 4 C T1015 Standard T1017 Large T1016 Screening Microplate assays per kit 400 1200 10 000 5X Dilution Buffer 5 mL 12087 15 mL T2090 125 mL 12280 Assay Buffer 20 mL 60 mL 500 mL CSPD Substrate 1 0 mL 3 0 mL 25 mL Reaction Buffer Diluent 19 mL 57 mL 475 mL Control Enzyme 50 uL 50 uL 425 uL is See Protocol Note 3 PROTOCOL REVISION for achieving
6. 20 min 9 Place plate in luminometer and measure for 0 1 1 sec well C Extract Preparation for Non Secreted Placental Alkaline Phosphatase This procedure is for adherent cells For non adherent cells please see Protocol Note 2 For the following hazards see the complete safety alert descriptions in Appendix C Safety on page 8 A N WARNING CHEMICAL HAZARDS 5X Dilution Buffer 1 Prepare a lysis buffer by diluting 5X Dilution Buffer to 1X 250 uL lysis buffer per 60 mm plate with H2O Add Triton X 100 not supplied to a final concentration of 0 2 v v 2 Rinse cells twice with PBS add lysis buffer and detach from plate with a cell scraper 3 Prepare extract by repeated pipetting and transfer to a microfuge tube Centrifuge for 2 min to pellet debris 4 Transfer extracts supernatant to a fresh tube Use immediately or store at 70 C 5 Aliquot 30 uL of cell extract into a microfuge tube and add 370 uL of 1X Dilution Buffer for tube assays or use 15 uL of extract with 185 uL of 1X Dilution Buffer for microplate assays 6 Proceed with the appropriate Detection Protocol Section III A or B starting at step 5 D Direct Lysis Procedure for Microplate Cultures This procedure is for adherent cells which express non secreted placental alkaline phosphatase cultured in 96 well tissue culture treated luminometer plates Heat inactivation is not effective with this protocol For the following hazards see t
7. EEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES Literature Citation When describing a procedure for publication using this product please refer to it as the Phospha Light System Trademarks Applied Biosystems AB Design CSPD and Tropix are registered trademarks and Emerald and Phospha Light are trademarks of Applied Biosystems Inc 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 Inc All rights reserved PREFACE Safety Information Note For general safety information see this Preface and Appendix C Safety on page 8 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 al
8. 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 5 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 AN WARNING CHEMICAL HAZARD 5X Dilution Buffer 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 Assay Buffer may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 11 WARNING CHEMICAL HAZARD Control Enzyme may cause eye skin and respiratory tract irritation Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves AN WARNING CHEMICAL HAZARD CSPD Substrate may cause eye skin and respiratory V 10 11 12 13 14 15 16 17
9. ay Buffer 100 uL tube and Reaction Buffer to room temperature Dilute sufficient 5X Dilution Buffer to 1X 100 300 uL sample with H20 Prepare a sample by diluting 100 uL of culture medium with 100 300 uL of 1X Dilution Buffer in a microfuge tube see Note 3 Heat at 65 C for 30 min then cool on ice to room temperature see Note 1 Add 100 ul of diluted sample to a luminometer tube Add 100 ul of Assay Buffer per tube and incubate for 5 min Add 100 ul of Reaction Buffer per tube and incubate for 20 min Place tubes in a luminometer and measure for 0 1 1 sec tube B Detection with Microplate Luminometers For the following hazards see the complete safety alert descriptions in Appendix C Safety on page 8 IN WARNING CHEMICAL HAZARDS CSPD Substrate Dilution Buffer Assay Buffer 1 Dilute sufficient CSPD substrate 1 20 with Reaction Buffer Diluent to make Reaction Buffer 50 uL well 2 Equilibrate Assay Buffer 50 uL well and Reaction Buffer to room temperature 3 Dilute sufficient 5X Dilution Buffer to 1X 50 150 uL sample with H20 4 Prepare a sample by diluting 50 uL of culture medium with 50 150 uL of 1X Dilution Buffer in a microfuge tube see Note 3 5 Heat at 65 C for 30 min then cool on ice to room temperature see Note 1 6 Add 50 wl of diluted sample to microplate wells 7 Add 50 ul of Assay Buffer per well and incubate for 5 min 8 Add 50 wl of Reaction Buffer per well and incubate for
10. d 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 9 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 2 MSDSs About MSDSs Chemical manufacturers supply current Material Safety 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 Bios
11. dia SEAP is a reporter protein that is secreted into the cell culture media and detected by testing aliquots of media leaving cells intact for further experimentation 1 2 SEAP is a truncated form of human placental alkaline phosphatase PLAP Detection of non secreted placental alkaline phosphatase is also possible see section III C and D The Phospha Light reporter gene assay incorporates CSPD chemiluminescent substrate and Emerald luminescence enhancer for high sensitivity and wide dynamic range 3 4 Phospha Light system has been used for detection of secreted placental alkaline phosphatase reporter enzyme in cell culture media 5 6 and for quantitation of non secreted placental alkaline phosphatase in both cell and tissue extracts 7 8 The Phospha Light detection assay is simple and rapid Secreted placental alkaline phosphatase is measured from 48 to 72 hours after cell transfection 2 Cell culture medium or cell lysate is incubated first with a buffer system that differentially inhibits non placental alkaline phosphatase serum and endogenous cellular alkaline phosphatase and then with CSPD containing Reaction Buffer until maximum light emission is reached approximately 20 minutes The light emission kinetics provide a persistent glow signal that enable measurement over a wide time interval Light signal output is measured in a luminometer without the need for automated injection capability Chemiluminescent reporter assays for
12. ed Biosystems Technical Support for additional questions C Safety 1 GENERAL CHEMICAL SAFETY Chemical hazard warning AS AAS AN ZN 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 bottles 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 an
13. ert 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 situation 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 MSDSs on page 9 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 l INTRODUCTION The Tropix Phospha Light system is a chemiluminescent reporter gene assay system designed for the rapid and sensitive detection of secreted placental alkaline phosphatase SEAP in cell culture me
14. he complete safety alert descriptions in Appendix C Safety on page 8 WARNING CHEMICAL HAZARDS CSPD Substrate 5X Dilution Buffer Assay Buffer a Dilute sufficient CSPD substrate 1 20 with Reaction Buffer Diluent to make Reaction Buffer 50 uL well 2 Prepare a lysis buffer by diluting 5X Dilution Buffer to 1X 10 uL lysis buffer per well with H20 Add Triton X 100 not supplied to a final concentration of 0 2 v v Dilute additional 5X Dilution Buffer to 1X 40 uL per well with H20 3 Rinse wells once with PBS 4 Add 10 uL of lysis buffer per well and incubate for 10 min 5 Add 40 uL of 1X Dilution Buffer per well 5 Add 50 uL of Assay Buffer per well and incubate for 5 min 6 Add 50 uL of Reaction Buffer per well and incubate for 20 min 7 Place plate in luminometer and measure for 0 1 1 sec well E Protocol Notes 1 Adding Assay Buffer to warm culture media or heating culture media with Assay Buffer may result in decreased sensitivity due to increased background Eliminating or decreasing the incubation time in Assay Buffer may have the same effect since non PLAP background activity will not inhibited to same extent 2 Non adherent cells may be pelleted and sufficient 1X Dilution Buffer 0 2 Triton X 100 added to cover the cells Cells can be resuspended and lysed by repeated pipetting 3 PROTOCOL REVISION In order to perform the assay on the maximum number of samples indicated as microplate assays k
15. i M X Tong A Skelton R Chase T Chen A Prongay SL Bogen AK Saksena FG Njoroge RL Veselenak RB Pyles N Bourne BA Malcolm and SM Lemon 2006 Mutations conferring resistance to SCH6 a novel hepatitis C virus NS3 4A protease inhibitor Reduced RNA replication fitness and partial rescue by second site mutations J Biol Chem 281 12 8205 8215 Hwang D R Y C Tsai J C Lee K K Huang R K Lin C H Ho J M Chiou Y T Lin JTA Hsu and C T Yeh 2004 Inhibition of Hepatitis C virus replication by arsenic trioxide Antimicrob Agents Chemother 48 8 2876 2882 Brandt S T Grunwald S Lucke A Stang and K Uberla 2006 Functional replacement of the R region of simian immunodeficiency virus based vectors by heterologous elements J Gen Virol 87 2297 2307 Alexander L PO Illyinskii SM Lang RE Means J Lifson K Mansfield and RC Desrosiers 2003 Determinanants of increased replicative capacity of serially passaged Simian Immunodeficiency Virus with nef deleted in Rhesus monkeys J Virol 77 12 6823 6835 Johnson WE J Morgan J Reitter BA Puffer S Czajak RW Doms and RC Desrosiers 2002 A replication competent neutralization sensitive variant of Simian Immunodeficiency Virus lacking 100 amino acids of envelope J Virol 76 5 2075 2086 12 20 21 22 23 24 25 26 27 28 29 30 31 Pohlmann S M Krumbiegel and F Kirchhoff 1999 Coreceptor usage of BOB GPR15 and Bonzo STRL33 by primary isolates
16. indicated capacity with reagent volumes provided 5X Dilution Buffer T2087 T2090 T2280 is also available separately if additional volume is desired 1 5X Dilution Buffer dilute to 1X with deionized H20 Phospha Light Assay Buffer contains a proprietary mixture of non placental alkaline phosphatase inhibitors 3 CSPD Chemiluminescent Substrate dilute in Phospha Light Reaction Buffer Diluent 4 Phospha Light Reaction Buffer Diluent contains Emerald luminescence enhancer 5 Control Enzyme purified human placental alkaline phosphatase 0 3 ng L in 150 mM Tris pH 7 8 50 mM NaCl 50 glycerol SEAP reporter vectors The Phospha Light assay system is NOT provided with SEAP reporter expression vectors These are available through various commercial suppliers for both cell culture transfection as well as in vivo delivery and expression Hl DETECTION PROTOCOL FOR SECRETED PLACENTAL ALKALINE PHOSPHATASE Please read the entire Protocol and Notes sections before proceeding Perform all assays in triplicate at room temperature unless otherwise indicated A Detection with Tube Luminometers For the following hazards see the complete safety alert descriptions in Appendix C Safety on page 8 WARNING CHEMICAL HAZARDS CSPD Substrate Dilution Buffer Assay Buffer 1 Dilute sufficient CSPD substrate 1 20 with Reaction Buffer Diluent to make Reaction Buffer 100 uL tube Equilibrate Ass
17. it in the table on page 3 use the smaller volume of 1X Dilution Buffer indicated and follow rest of protocol as indicated ie prepare 50 uL of 1X Dilution Buffer per sample in Section B Step 3 and mix 50 uL of 1X Dilution Buffer with culture medium in Step 4 If using the smaller volume of 1X Dilution Buffer new recommendation samples will be more concentrated than previous recommendation maximum volume indicated Therefore it may be possible or more ideal to use a smaller volume of the original culture medium sample IV APPENDICES A Preparation of Controls Positive Control For the following hazards see the complete safety alert descriptions in Appendix C Safety on page 8 I WARNING CHEMICAL HAZARDS Control Enzyme Dilution Buffer The stock enzyme supplied is approximately 0 3 ng uL 0 75 U mL Generate a standard curve by serially diluting the stock enzyme in 1X Dilution Buffer or mock transfected cell culture media A 10 UL aliquot of the stock enzyme undiluted should be used for the high end detection limit 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 Additi
18. of human immunodeficiency virus type 1 J Gen Virol 80 1241 1251 Rubenstrunk A C Orsini A Mahfoudi and D Scherman 2003 Transcriptional activation of the metallothionein gene by electric pulses in vivo Basis for the development of a new gene switch system J Gene Med 5 773 783 Riera M M Chillon JM Aran JM Cruzado J Torras JM Grinyo and C Fillat 2004 Intramuscular SP1017 formulated DNA electrotransfer enhances transgene expression and distributes hHGF to different rat tissues J Gene Med 6 111 118 Duboise M J Guo S Czajak H Lee R Veazey RC Desrosiers and JU Jung 1998 A role for Herpesvirus Saimiri orf14 in transformation and persistent infection J Virol 72 8 6770 6776 Latta Mahieu M M Rolland C Caillet M Wang P Kennel Mahfouz Loquet J F Dedieu A Mahfoudi E Trannoy and V Thuillier 2002 Gene transfer of a chimeric trans activator is immunogenic and results in short lived transgene expression Human Gene Therapy 13 1611 1620 Khan AS LC Smith RV Abruzzese KK Cummings MA Pope PA Brown and R Draghia Akli 2003 Optimization of electroporation parameters for the intramuscular delivery of plasmids in pigs DNA Cell Biol 22 12 807 814 Zhao H and BPH Peeters 2003 Recombinant Newcastle Disease Virus as a viral vector Effect of genomic location of foreign gene on gene expression and virus replication J Gen Virol 84 781 788 Zabeau L D Defeu J Van der Heyden H Iserentant J Vandekerckhove and J Tave
19. onal positive controls can include use of control SEAP constructs that provide constitutive expression of reporter enzyme as a positive control for cell transfection Alternatively stock enzyme can be prepared by reconstituting lyophilized human placental alkaline phosphatase Sigma P 3895 to 1 mg mL in 1X Dilution Buffer containing 0 1 BSA and 50 glycerol Store at 20 C Negative Control Assay a volume of culture media from mock transfected cells equivalent to that of experimental cell culture media used to determine endogenous cellular 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 insturment set for luminescence measurement to measure the light emission from 96 or 384 well microplates The linear range of detection will vary according to cell type and on the reporter enzyme expression level The number of cells or sample volume 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 and use the positive control serial dilution curve to determine the upper limit for your specific luminometer Contact Appli
20. ons 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 10 4 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 containment 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
21. rnier 2004 Functional analysis of leptin receptor activation using a Janus kinase signal transducer and activator of transcription complementation assay Mol Endocrinol 18 1 150 161 Sakai J RB Rawson PJ Espenshade D Cheng AC Seegmiller JL Goldstein and MS Brown 1998 Molecular identification of the sterol regulated luminal protease that cleaves SREBPs and controls lipid composition of animal cells Mol Cell 2 505 514 Kagan JC M P Stein M Pypaert and CR Roy 2004 Legionella subvert the functions of Rab1 and Sec22b to create a replicative organelle J Exp Med 199 9 1201 1211 Arunachalam L L Han NG Tassew Y He L Wang L Xie Y Fujita E Kwan B Davletov PP Monnier HY Gaisano and S Sugita 2008 Munc18 1 is critical for plasma membrane localization of Syntaxin1 but not of SNAP 25 in PC12 cells Mol Biol Cell 19 722 734 Bossard C D Bresson RS Polishchuk and V Malhotra 2007 Dimeric PKD regulates membrane fission to form transport carriers at the TGN J Cell Biol 179 6 1123 1131 For complete updated reference list AB 120MI09 please see http www appliedbiosystems com Product amp Service Literature 13
22. secreted placental alkaline phosphatase may be conducted in cells that have endogenous non placental alkaline phosphatase activity Endogenous non placental enzyme activity is significantly reduced with a combination of heat inactivation and differential inhibitors that do not significantly inhibit the transfected placental alkaline phosphatase It is important to determine the level of endogenous enzyme in media of non transfected cells in order to establish assay background Certain cell lines such as HeLa and others derived from cervical cancers may express placental alkaline phosphatase which may produce high assay backgrounds when shed into the media 9 Therefore the use of secreted alkaline phosphatase as a reporter system in these cell lines is generally not recommended Applications The Phospha Light reporter gene assay system has been used widely for reporter gene assays to measure gene expression in established cell lines 10 and in transfected primary cells 11 12 including as a gene knockdown RNA interference read out 13 The Phospha Light reporter gene assay has been used for a wide variety of viral functional assays including viral gene expression assays 14 15 viral replication 16 17 viral fusogenicity 18 virus neutralization and viral mediated cell cell fusion 19 and viral infectivity 20 The SEAP reporter protein is very enabling for in vivo reporter gene assays by assaying serum samples from transgenic transfected
23. ystems 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 3 CHEMICAL WASTE SAFETY Chemical waste hazards CAUTION HAZARDOUS WASTE Refer to Material Safety Data Sheets and local regulations for handling and disposal AN WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death A 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
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