PathHunter® eXpress β-Arrestin Orphan GPCR Assays
Contents
1. MATERIALS at room temperature 23 C DO NOT pipette up and down in the well to mix or vortex shake plates The following products are recommended 3 Read samples on any standard luminescence plate reader l 9 LDH Quantification Kit DiscoveRx Cat 92 2002 Use GraphPad Prism or other comparable program to plot your agonist dose 9 CytoTracker Glutathione Quantification Kit DiscoveRx Cat 92 2003 response 9 DNA Damage Quantification Kit DiscoveRx Cat 92 2004M Products not available in all countries Please inquire ASSAY INCUBATION AND CELL PLATING REAGENT REQUIREMENTS PathHunter eXpress B Arrestin Orphan GPCR Assay can be validated for optimal assay performance at either 24 or 48 hours post thaw Always use the CP Reagent included in the kit and DO NOT substitute from an alternate kit at any time 10 7 SOLVENTS AND PREPARATION OF COMPOUND DILUTIONS PathHunter eXpress B Arrestin Orphan GPCR assays should be carried out in the presence of lt 1 solvent i e DMSO ethanol PBS or other As solvents can affect assay performance optimize the assay conditions accordingly if other solvents or solvent concentrations are required For preparation of test compounds we recommend preparing the dilutions using the Cell Plating Reagent provided in the kit containing the appropriate solvent For antibodies or other compounds that may be sensitive to serum2. 803 _ Figure 2 Cell lysates prepared from PathHunter eXpress B Arrestin Orphan GPCR cells were treated with PNGase F Glyko Cat GKE 5003 run on a SDS PAGE gel and analyzed using the EAstern Blot Assay Kit DiscoveRx Cat 93 0053 Untreated lane resolves a band of appro priate size corresponding to GPCR PK fusion protein and the PNGase F treated lane resolves a deglycosylated band indicative of proper expression and folding of GPCR protein 30 000 Without EA m With EA 22497 25 000 20 000 1 D 45 000 40 000 1 5 000 1 EA Arrestin Parental GPR6 Figure 3 PathHunter eXpress B Arrestin Orphan GPCR cells were analyzed for basal activity as well as GPCR ProLink expression by comparing the ratio of signal between untreated cells and cells treated with saturating amounts of exogenous EA using ProLink Detection Kit DiscoveRx Cat 92 0006 Signal from complementation of ProLink and EA fragments correlates to the amount of GPCR PK expression in the cell line Figure 4 Viability of PathHunter eXpress B Arrestin Orphan GPCR cells was confirmed by bright field microscopy KIT CONTENTS AND STORAGE CONDITIONS PATHHUNTER EXPRESS KIT COMPONENTS REQUIRE MULTIPLE STORAGE TEMPERATURES OPEN BOXES IMMEDIATELY AND STORE CONTENTS AS INSTRUCTED SHELF LIFE Use kit within 6 months from the date of receipt under proper storage conditions Box 1 PATHHUNTER EXPRESS ARRESTIN ORPHAN GPCR CEL
3. Contact Information DiscoveRx Corporation World Wide Headquarters 42501 Albrae Street Fremont CA 94538 United States t 1 510 979 1415 f 1 510 979 1650 toll free 1 866 448 4864 DiscoveRx Corporation Ltd Europe Headquarters Faraday Wharf Holt Street Birmingham Science Park Aston Birmingham B7 4BB United Kingdom t 44 121 260 6142 f 44 121 260 6143 KINOMEscan A division of DiscoveRx 11180 Roselle Street Suite D San Diego CA 92121 United States t 1 800 644 5687 f 1 858 630 4600 BioSeek A division of DiscoveRx 310 Utah Avenue Suite 100 South San Francisco CA 94080 United States t 1 650 416 7600 f 1 650 416 7625 www discoverx com Discover 2014 DiscoveRx Corporation Fremont CA 94538 70 256 DRx_UM_PHeX_ORPHAN_0914V6 All rights reserved Discover PathHunter C erress PathHunter eXpress B Arrestin Orphan GPCR Assays User Manual Simple Solutions for Complex Biology CONTENTS LEGAL SECTION INTENDED USE TECHNOLOGY PRINCIPLE CHARACTERIZATION OF ORPHAN GPCR CELLS KIT CONTENTS AND STORAGE CONDITIONS RECOMMENDED MATERIALS MATERIALS PROVIDED ADDITIONAL MATERIALS REQUIRED NOT PROVIDED ASSAY INCUBATION AND CELL PLATING REAGENT REQUIREMENTS SOLVENTS AND PREPARATION OF COMPOUND DILUTIONS USE OF PLASMA OR SERUM CONTAINING SAMPLES THAWING AND PLATING FROZEN CELLS ASSAY PROCEDURE AGONIST DOSE RESPONSE PLATE MAP PROTOCOL Q
4. LS STORAGE Short term 2 weeks or less Store vials at 80 C immediately upon arrival Long term greater than 2 weeks Place vials in the vapor phase of liquid nitrogen N2 PathHunter eXpress cells arrive frozen on dry ice Cells are delivered in individual vials containing 1 x 10 cells in 100 uL of freezing medium Each vial contains sufficient cell numbers to generate 1 96 well microplate prepared at the seeding density described When removing cryovials from liquid N storage use tongs and place immediately on dry ice in a covered container Wait at least one minute for any liquid Nzinside the vial to evaporate and proceed with the thawing protocol page 9 Do not touch the bottom of the tubes at any time to avoid inad vertent thawing of the cells If cells are not frozen upon arrival do not proceed Contact technical support Box 2 PATHHUNTER DETECTION REAGENTS AND CP REAGENT Store at 20 C Once thawed store the Cell Plating CP Reagent at 4 C Avoid multiple freeze thaw cycles In rare instances the CP Reagent may be yellow in color after thaw ing Although this indicates a slight change in pH continue with the assay as this does not impact assay performance Thaw the PathHunter Detection Reagents at room temperature before use and after thawing store reagents for up to 7 days at 4 C The reagents can tolerate up to three freeze thaw cycles with no impact on performance Once made the work ing solution is st
5. UICK START PROCEDURE FREQUENTLY ASKED QUESTIONS APPENDIX A RELATED PRODUCTS PAGE 3 PAGE 4 PAGE 4 PAGE 5 PAGE 6 PAGE 6 PAGE 7 PAGE 7 PAGE 7 PAGE 8 PAGE 8 PAGE 8 PAGE 9 PAGE 9 PAGE 11 PAGE 12 PAGE 14 NOTES 15 APPENDIX RELATED PRODUCTS Cell Plating Reagents 93 0563R Series PathHunter eXpress B Arrestin Many GPCR Assays PathHunter eXpress B Arrestin Many Orphan Assays PathHunter eXpress B Arrestin Many Ortholog GPCR Assays For additional information or technical support please call 1 866 448 4864 US For more information visit www discoverx com certified cell_plating_reagents php www discoverx com gpcrs express_arrestin php www discoverx com gpcrs express_orphan php www discoverx com gpcrs express_ortholog php or 44 121 260 6142 Europe or email info discoverx com 14 LEGAL SECTION This product and or its use is covered by one or more of the following U S patents 6 342 345 84 7 135 325 52 8 101 373 52 and or foreign patents patent applications and trade secrets that are either owned by or licensed to DiscoveRx Corporation LIMITED USE LICENSE AGREEMENT The cells and detection reagents collectively Materials purchased from DiscoveRx are expressly restricted in their use DiscoveRx has developed a Protein Protein Interaction assay Assay that employs genetically modified cells and vectors collectively the Cells an
6. able for 24 hours at room temperature Box 3 96 WELL TISSUE CULTURE TREATED PLATE Store at Room Temperature QUICK START PROCEDURE AGONIST DOSE RESPONSE Plate 100 uL PathHunter eXpress cells well Incubate 24 or 48 hours 37 C Add 10 uL of agonist Incubate 90 minutes 37 C Add 55 uL Detection Reagent Working Solution Incubate 60 Minutes Room Temperature Read Chemiluminescent Signal 11 Remove 30 uL of diluted compound from tube 12 add it to tube 11 and MATERIALS PROVIDED mix gently by pipetting up and down Discard the pipet tip Each shipment contains f With a clean pipet tip remove 30 uL of diluted compound from tube 11 add it to tube 10 and mix gently by pipetting up and down Discard the Description Contents Contents Contents Storage pipet tip Box 1 1 vial 2 vials 10 vials 80 C short 6 6 6 i g Repeat this process 8 more times preparing serial dilutions from right to ae ne lt Dr eee PO ENE GEIE ER ae GEIS ea kiquid N B Arrestin Orphan GPCR long left across the tubes Cells h DO NOT add agonist compound to tube 1 This sample serves as the Boe D no agonist control and completes the dose curve Detection 100 dp 200 dp 1 000 dp i Repeat this process for each compound to be tested Reagents j Set compounds aside until they are ready to be added Cell Assay Buffer REME 2a mE mL j p Subs
7. and or other assay components dilutions can be prepared in either Hanks Buffered Salt Solution HBSS 10 mM HEPES 0 1 Bovine Serum Albumin BSA or OptiMEM 0 1 BSA without affecting assay performance USE OF PLASMA OR SERUM CONTAINING SAMPLES PathHunter eXpress B Arrestin Orphan GPCR assays can be run in the presence of high levels of serum or plasma without negatively impacting assay performance Standard curves of control ligand can be prepared in neat heparinized plasma and added directly to the cells without further dilution ie 100 plasma in the well After ligand stimulation the samples should be removed and replaced with fresh Cell Plating Reagent before the addition of the PathHunter Detection Reagents NOTE EDTA anti coagulated plasma samples do not give a positive response in the assay Therefore the choice of anti coagulant treatment is very important THAWING AND PLATING FROZEN CELLS The following steps outline the procedure for thawing and plating frozen PathHunter eXpress cells from freezer vials 1 Pre warm the CP reagent in a 37 C water bath 2 Remove cell vial s from 80 C or liquid N2 vapor phase storage and place immediately on dry ice prior to thawing DO NOT EXPOSE VIALS TO ROOM TEMPERATURE NOTE When removing cryovials from liquid Nz place immediately on dry ice in a covered container Wait at least one minute before opening for any liquid N inside the vial to evaporate 3 Place the cel
8. cific expiration date information What if my CP reagent changes from a red pink color to yellow after freezing thawing If the CP reagent changes color from red pink to yellow after thawing please continue with the assay according to the product insert We have observed this color change on rare occasions and have confirmed that it does not affect assay performance Can I use the PathHunter eXpress Arrestin Orphan GPCR assay to test inverse agonist compounds Yes For compounds that behave as inverse agonists follow the agonist protocol as described on page 9 Can the source of my agonist compound impact my assay performance Yes The vendor source of compound can impact assay performance dramati cally Compounds can vary in purity from vendor to vendor In addition vendors will recommend different diluents methanol NaOH ethanol DMSO water different treatments boiling freeze thaw etc or different storage temperatures for the same compound Can I use this assay to test human plasma or serum samples Yes PathHunter eXpress B Arrestin Orphan GPCR assays tolerate up to 80 serum or plasma First prepare a standard curve of spiked ligand in neat heparinized plasma or mouse human serum Add samples directly to the cells no further dilution 100 plasma in the well After stimulation remove the plasma or serum sample and replace with fresh CP reagent before addition o f the PathHunter Detection Reagents It has been show
9. compound in CP Reagent containing the appropriate solvent DMSO ethanol PBS or other The concentration of each dilution should be prepared at 11X of the final screening concentration i e 10 uL compound 100 uL of cells For each dilution the final concentration of solvent should remain constant Preparation of 12 point dose curve serial dilutions a Label tubes 1 through 12 b Add 60 uL of Cell Plating Reagent containing appropriate solvent to tubes 1 11 Prepare a working concentration of agonist compound in CP Reagent d Add 90 uL of the working concentration of agonist compound to tube 12 9
10. d related detection reagents the Reagents collectively referred to as Materials By purchasing and using the Materials the Purchaser agrees to comply with the following terms and conditions of this label license and recognizes and agrees to such restrictions 1 The Materials are not transferable and will be used only at the site for which they were purchased Transfer to another site owned by Purchaser will be permitted only upon written request by Purchaser followed by subsequent written approval by DiscoveRx 2 The Reagents contain or are based upon the proprietary and valuable know how developed by DiscoveRx and the Reagents have been optimized by DiscoveRx to function more effectively with the Cells in performing the Assay Purchaser will not analyze or reverse engineer the Materials nor have them analyzed on Purchaser s behalf 3 In performing the Assay Purchaser will use only Reagents supplied by DiscoveRx or an authorized DiscoveRx distributor for the Materials If the purchaser is not willing to accept the limitations of this limited use statement and or has any further questions regarding the rights conferred with purchase of the Materials please contact Licensing Department DiscoveRx Corporation 42501 Albrae Street Fremont CA 94538 USA tel 1 510 979 1415 x104 info discoverx com For some products cell lines certain 3 party gene specific patents may be required to use the cell line It is the purcha
11. e signal stable for The signal is stable for 24 hours after addition of detection reagent My cells are floating after the 48 hours incubation The cells are not viable contact technical support for a replacement Can I switch plates or should I use the plate provided You can use any clear bottom white or opaque walled plate What if cells are not completely adherent after 24 48 hrs incubation For certain targets cells may not be completely adherent after 24 hours but still greater than 80 viable Please continue on with the protocol as described in the product insert Why do longer incubation times with Detection Reagents lead to a higher signal The complemented B galactosidase B gal enzyme is continually turning over the substrate Theoretically the signal continues to increase until the substrate is exhausted Therefore the longer you incubate the reaction the higher the RLU values 12 CHARACTERIZATION OF ORPHAN GPCR CELLS PathHunter eXpress B Arrestin Orphan GPCR cells are validated using the following criteria a Confirmation of proper GPCR expression at the predicted molecular weight Figure 2 b In vitro complementation studies to measure basal activity and GPCR PK expression Figure 3 and c Cell viability after overnight incubation in the appropriate Cell Plating Reagent Figure 4 1 2 3 98kDa _ Lane 4 MW Markers Lane 2 no PNGase F Lane 3 with PNGaseF 51808 9 39kDa_ ga lt GPR17 ym 19
12. l vial s briefly 10 seconds to 1 min in a 37 C water bath until only small ice crystals remain and the cell pellet s is almost completely thawed 4 Add 0 5 mL of pre warmed CP reagent to the cell vial Pipette up and down gently several times to ensure that the cells are evenly distributed 5 Immediately transfer the cells to 11 5 mL of pre warmed CP reagent and pour into a disposable reagent reservoir 6 Plate 100 uL of cells into each well of the provided 96 well tissue culture plate Ir 6 M O A gt 7 After seeding the cells into the microplate incubate for either 24 or 48 hours at 37 C 5 0 ASSAY PROCEDURE AGONIST DOSE RESPONSE The steps outlined below provide the assay volumes and procedure for performing agonist assays using the PathHunter eXpress B Arrestin Orphan GPCR cells and De tection Reagents Although plate layouts and experimental designs may vary we recommend performing a 12 point dose curve for each compound using at least duplicate wells for each dilution The protocol and volumes described below are designed for a complete 96 well plate D O 3 fold serial dilution of agonist 1 2 3 4 5 6 7 8 9 10 11 12 Compound 1 Compound 2 Compound 3 Compound 4 DAY 2 or 3 AGONIST COMPOUND PREPARATION AND ADDITION 1 Dissolve agonist compound in the vehicle of choice DMSO Ethanol PBS or other at the desired concentration 2 Prepare 3 fold serial dilutions of agonist
13. n that EDTA anti coagulated plasma inhibits EFC and should be avoided for these types of studies Can I use the PathHunter eXpress f Arrestin Orphan GPCR Assays to study novel GPCR Heterodimers Yes To perform a heterodimer experiment transiently transfect the PathHunter eXpress cells with a plasmid expressing an untagged GPCR receptor Twenty four hours post transfection follow the agonist protocol as described on page 9 and treat the cells with ligand specific to the untagged GPCR and measure the effects on the PK tagged GPCR using PathHunter Detection Reagents As a negative control for the assay use cells expressing the single GPCR to deter mine selectively and specificity of your compounds For these experiments we recommend use of a lipid based transfection reagent such as FUGENE 6 Transfection Reagent Roche Applied Science Cat 11 815 091 001 Visit www discoverx com gpcrs dimerization for more details Can my eXpress assay be run in 384 well format All PathHunter eXpress assays are optimized and formatted to be run in 96 well plates Certain assays also perform well in 384 well format but require modifi cations to the protocol cell numbers incubation times etc Please contact Technical Support techsupport discoverx com for more information 13 FREQUENTLY ASKED QUESTIONS Q A PO rO PO FO FO gt lt Confirm that the final ligand concentration is correct Some ligands are
14. rotein of B Arrestin and the larger N terminal deletion mutant of B gal called enzyme acceptor or EA Activation of the GPCR stimulates binding of B Arrestin EA to the ProLink tagged GPCR and forc es complementation of the two enzyme fragments resulting in the formation of an active B gal enzyme This action leads to an increase in enzyme activity that can be measured using chemiluminescent PathHunter Detection Reagents Because Arrestin recruitment occurs independent of G protein coupling these assays provide a direct universal platform for measuring receptor activation GPCR Tagged with ProLink Stimulation amp Translocation p Arrestin EA A Substrate Light B Gal EA Acceptor B Arrestin EA Inactive beta gal Figure 1 PathHunter f Arrestin Assay Principle Activation of the ProLink tagged GPCR results in B Arrestin recruitment and formation of a functional enzyme capable of hydrolyzing substrate and generating a chemiluminescent signal A FREQUENTLY ASKED QUESTIONS CONTINUED Q A gt gt O gt What 15 the shelf life of the eXpress kits We recommend that eXpress kits should be used within 6 months of receipt under proper storage conditions For short term 2 weeks or less store eXpress cells at 80 C For long term storage more than 2 weeks store in the vapor phase of liquid nitrogen N2 Store the Detection Reagent Kit at 20 C Refer to the kit label for lot spe
15. ser s responsibility to determine if such patents or other intellectual property rights are required INTENDED USE PathHunter eXpress f Arrestin Orphan GPCR kits are ready to use complete kits that contain everything you need to perform a functional whole cell GPCR assay in live cells but without any cell culture The eXpress kits include single use vials of frozen cells stably expressing the Orphan GPCR of interest optimized cell culture reagents chemiluminescent detection reagents and plates Simply thaw and plate the pre validated cells and challenge with compound 24 or 48 hours later Whether you are measuring one or multiple GPCR responses to compound chal lenge the ready to assay eXpress format eliminates the need for lengthy expen sive and time consuming cell culture and makes functional testing fast and conven ient Assays are designed for 96 well plate analyses and kits include enough cells and detection reagents for either 100 data points 200 or 1000 data points Test compounds are not included and must be provided by the researcher TECHNOLOGY PRINCIPLE PathHunter B Arrestin products monitor GPCR activity by detecting the interaction of B Arrestin with the activated GPCR using B galactosidase 98 enzyme fragment complementation EFC Figure 1 In this system the GPCR of interest is fused in frame with the small 42 amino acid fragment of B gal called ProLink and co expressed in cells stably expressing a fusion p
16. sticky I did not see a signal with my control agonist There may be differences in agonist purchased from different vendors Confirm that the control agonist used is the same ligand used in the dose response shown in the provided cell specific datasheet I did not see a response with my compound The concentration of DMSO or Ethanol used for dilution is too high Maintain concentration of the agonist antagonist diluent at lt 1 and difficult to dissolve My cells arrived thawed Can I use them No Call technical support for a replacement How long is the prepared detection reagent good for The working detection reagent solution must be used within 8 hours of mixing What instruments can I use to read the plates Any bench top luminometer will work with the PathHunter eXpress Arrestin Orphan GPCR Assays Below is a partial list of commercially available luminom eters that have been used to validate our assays Turner Biosystems Modulus Microplate GE Healthcare Life Sciences LEADseeker FarCyte BMG Labtech PHERAstar Plus LUMIstar Omega Perkin Elmer TopCount VICTOR II or V Fusion LumiCount EnVision MicroBeta Trilux ViewLux Molecular Devices CLIPR LJL Acquest LJL Analyst LJL Analyst HT LJL Analyst GT Gemini SpectraMax Flexstation LMax Tecan Ultra Evolution Beckman Coulter CRi Berthold Technologies Mithras LB 940 Hamamatsu FDSS6000 FDSS RayCatcher How long is th
17. trate Reagent 1 1 5 mL 2 5 mL 15 0 mL 3 Remove PathHunter eXpress cells previously plated on day 1 from the incubator Substrate Reagent 2 0 3 mL 0 5 mL 3 0 mL Cell Plating Reagent 1 x 20 mL 2 x 20 mL 2 x 100 mL 4 Transfer 10 uL from tubes 1 12 to duplicate wells according to the plate map 2 2 on p 9 Box 3 l 96 well Tissue Culture 1 plate 2 plates 10 plates Room Temp 5 Incubate for 90 minutes 37 C Treated Plate SUBSTRATE PREPARATION AND ADDITION Centrifuge vial before opening to maximize recovery Refer to cell line specific data sheets for optimized Cell Plating Reagent included with each kit 1 During the incubation period prepare a working stock of PathHunter Detection Reagents by mixing 19 parts Cell Assay Buffer 5 parts Substrate Reagent 1 and 1 part Substrate Reagent 2 Cell Assay Buffer 4 75 mL ADDITIONAL MATERIALS REQUIRED NOT PROVIDED The following additional materials are required but not provided Pipettes and pipette tips Tissue culture disposables GPCR Test compound s Disposable Reagent Reservoir such as Thermo Scientific Cat 8094 or similar 96 well V bottom compound dilution plates DiscoveRx Cat 92 0011 Multi mode or luminescence plate reader Substrate Reagent 1 1 25 mL Substrate Reagent 2 0 25 mL De E e ae NOTE The working solution is stable for up to 8 hours at room temperature 2 55 uL of prepared detection reagent per well and incubate for 60 minutes RECOMMENDED
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