PathHunter® eXpress Activated GPCR Internalization Assays
Contents
1. Incubate 30 minutes 37 C Tie ieee Add 5 uL of Agonist ECgo Incubate 3 hours 25 C or 37 C Add 55 pL Detection Reagent Working Solution Incubate 60 Minutes Room Temperature Ve eee Read Chemiluminescent Signal Please refer to the cell line specific datasheet for any variation in assay conditions 16 USE OF PLASMA OR SERUM CONTAINING SAMPLES PathHunter eXpress Activated GPCR Internalization Assay 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 hepa rinized 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 CP 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 Activated GPCR Internalization cells from freezer vials Pre warm 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 NOTE2. Compound 2 Compound 3 Compound 4 zro7m7mmo0n 8 gt 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 Prepare 3 fold serial dilutions of agonist 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 pL compound 100 ul of cells For each dilution the final concentration of solvent should remain constant Preparation of 12 point dose curve serial dilutions We recommend starting with a concentration that is 50X the expected ECs value for the compound 550X ECs would be the final screening concentration Example If the expected ECso is 10 nM prepare the highest starting concentration at 5 5 uM a For each compound tested label tubes 1 through 12 b Add 60 uL of CP Reagent to tubes 1 11 c Prepare a working concentration of agonist compound in appropriate CP Reagent d Add 90 uL of the working concentration of agonist compound to tube 12 e Remove 30 ul of diluted compound from tube 12 add it to tube 11 and mix gently by pipetting up and down Discard the pipet tip 10 SUBSTRATE PREPARATION AND ADDITION During the incubation period prepare a working stock of PathHunter Detection Reagents by mixing 19 parts Cell Assay Buffer 5 parts Substrate Reage
3. down in the well to mix or vortex shake plates 18 MATERIALS PROVIDED Description Box 1 PathHunter eXpress Activated GPCR Inter nalization Cells 1 vial 1x10 cells ea Contents 2 vials 1x10 cells ea Storage 10 vials 80 C short 1x10 cells ea Liquid N2 long Box 2 PathHunter Detection 100dp 200 dp 1000 dp Reagents Cell Assay Buffer 5 7 mL 9 5 mL 57 0 mL 20 C Substrate Reagent 1 1 5 mL 2 5 mL 15 0 mL Substrate Reagent 2 0 3 mL 0 5 mL 3 0 mL Cell Plating Reagent 1 X 20 0 mL 2 X 20 0 mL 2 X 100 mL Box 3 96 well Tissue Culture 1 plate 2 plates 10 plates Room Temp Treated Plates Refer to cell line specific data sheets for optimized Cell Plating Reagent included with each kit Centrifuge vial before opening to maximize recovery ADDITIONAL MATERIALS REQUIRED NOT PROVIDED The following additional materials are required but not provided 1 Pipettes and pipette tips 2 Tissue culture disposables 3 GPCR control agonist as recommended in the cell line specific datasheet Visit www discoverx com pathway_assays control_ligands php for the complete DiscoveRx offering 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 Bie V e RECOMMENDED MATERIALS The following products are recommended e C
4. 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 70 261 DRx_UM_PHeX_ACTIVE_0914V5 All rights reserved DiscoveR Mes PathHunter eXpress Activated GPCR Internalization Assays User Manual Simple Solutions for Complex Biology CONTENTS LEGAL SECTION INTENDED USE TECHNOLOGY PRINCIPLE PROTOCOL OVERVIEW KIT CONTENTS AND STORAGE CONDITIONS MATERIALS PROVIDED ADDITIONAL MATERIALS REQUIRED NOT PROVIDED RECOMMENDED MATERIALS ASSAY INCUBATION AND MEDIA REQUIREMENTS COMPOUND PREPARATION AND INCUBATION TEMPERATURES USE OF PLASMA OR SERUM CONTAINING SAMPLES THAWING AND PLATING FROZEN CELLS ASSAY PROCEDURE AGONIST DOSE RESPONSE PLATE MAP PROTOCOL QUICK START PROCEDURE ASSAY PROCEDURE ANTAGONIST
5. DOSE RESPONSE PLATE MAP PROTOCOL QUICK START PROCEDURE ASSAY PROCEDURE ALLOSTERIC MODULATOR DOSE RESPONSE PLATE MAP PROTOCOL QUICK START PROCEDURE FREQUENTLY ASKED QUESTIONS APPENDIX A RELATED PRODUCTS PAGE 3 PAGE 4 PAGE 4 PAGE 5 PAGE 6 PAGE 7 PAGE 7 PAGE 7 PAGE 8 PAGE 8 PAGE 9 PAGE 9 PAGE 10 PAGE 10 PAGE 12 PAGE 13 PAGE 13 PAGE 16 PAGE 17 PAGE 17 PAGE 19 PAGE 20 PAGE 23 APPENDIX A RELATED PRODUCTS Description Catalog Number For more information visit Control Ligands AssayComplete Cell Plating Reagents Many 93 0563ROA 93 0563R5A 93 0563R27A http www discoverx com pathway_assays control_ligands php http www discoverx com certified cell_plating_reagents php PathHunter eXpress B Arrestin Many www discoverx com GPCR Assays gpcrs express_arrestin php PathHunter eXpress B Arrestin Many www discoverx com Orphan GPCR Assays gpcrs express_orphan php PathHunter eXpress B Arrestin Many www discoverx com Ortholog GPCR Assays gpcrs express_ortholog php 23 FREQUENTLY ASKED QUESTIONS CONTINUED Q What is the length of compound incubation required for optimal detection of internalization and recycling of the receptor A Optimal compound incubation times are somewhat target specific However the majority of receptors plateau within 2 to 3 hours Therefore the PathHunter eXpress Activated GPCR Inte
6. E LICENSE AGREEMENT The designated cells and reagents purchased from DiscoveRx are restricted in their use DiscoveRx has developed an assay for translocation and internalization Assay employing genetically modified cells Cells and detection reagents Reagents collectively referred to as Materials The Cells and Reagents are designed and optimized to be used together in the Assay DiscoveRx wishes to ensure that these Cells and Reagents are used properly and effectively By purchasing the Materials you recognize and agree to the 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 Purchaser will not analyze the Reagents nor have them analyzed on Purchas er s behalf 3 Purchaser will use only the Reagents supplied by DiscoveRx or an authorized DiscoveRx distributor for the Assays 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 3rd party gene specific patents may be req
7. ERVIEW Please read the entire protocol completely before running the assay Successful results depend on performing these steps correctly Refer to the cell line specific datasheet for additional information on optimized cell plating reagent and reference ligand For additional information or Technical Support contact DiscoveRx or visit www discoverx com The following steps are required to monitor the fate of activated and internalized GPCRs using a PathHunter eXpress Activated GPCR Internalization assay Figure 2 1 Thaw and plate frozen assay ready eXpress cells page 9 2 Dilute and add compounds 3 Perform functional assay in agonist page 10 or antagonist mode page 13 Plate cells Incubate 24 or 48 hours amp at 37 C Add compounds Incubate 90 minutes at 37 C PathHunter Detection Reagents Incubate 60 minutes at room temperature Read Chemiluminescence using a standard plate reader F Figure 2 Monitor functional GPCR responses to compound challenge using the fast and simple PathHunter eXpress Activated GPCR Internalization assay protocol KIT CONTENTS AND STORAGE CONDITIONS PATHHUNTER EXPRESS ACTIVATED GPCR INTERNALIZATION KIT COMPO NENTS REQUIRE MULTIPLE STORAGE TEMPERATURES OPEN BOXES IMME DIATELY 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 ACTIVATED GPCR INTERNA
8. LIZATION CELLS STORAGE Short term 2 weeks or less Store vials 80 C immediately upon arrival Long term greater than 2 weeks Place vials in the vapor phase of liquid nitrogen N2 PathHunter eXpress Activated GPCR Internalization cells arrive frozen on dry ice Cells are delivered in individual vials containing 1x10 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 Nz inside 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 inadvertent thawing of the cells If cells are not frozen upon arrival do not proceed Contact technical support Box 2 PATHHUNTER DETECTION REAGENT 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 thawing 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 On
9. When removing cryovials from liquid N2 place immediately on dry ice in a covered container Wait at least one minute before opening for any liquid N2 inside the vial to evaporate 3 Place the cell 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 After seeding the cells into the microplate incubate for either 24 or 48 hours at 37 C 5 CO2 NOTE Please refer to the cell line specific datasheet for any variation in assay conditions ASSAY PROCEDURE AGONIST DOSE RESPONSE The steps outlined below provide the assay volumes and procedure for performing agonist assays using the PathHunter eXpress Activated GPCR Internalization cells and PathHunter Detection 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 ra 2 C o a e 3 fold serial dilution of agonist Compound 1
10. ce made the work ing solution is stable for 24 hours at room temperature Box 3 96 WELL TISSUE CULTURE TREATED PLATES Store at Room Temperature 3 Read samples on any standard luminescence plate reader 4 Use GraphPad Prism or other comparable program to plot your allosteric modulator dose response QUICK START PROCEDURE ALLOSTERIC MODULATOR DOSE RESPONSE Plate 100 uL PathHunter eXpress cells well Incubate 24 or 48 hours 37 C Add 5 uL of Allosteric Modulator Incubate 30 minutes l Add 5 uL of Agonist Incubate 3 hours 25 C 37 C Add 55 uL Detection Reagent Working Solution Incubate 60 Minutes Room Temperature Read Chemiluminescent Signal Please refer to the cell line specific datasheet for any variation in assay conditions 19 5 e Remove 30 uL of diluted compound from tube 12 add it to tube 11 and mix gently by pipetting up and down Discard the pipet tip f With a clean pipet tip remove 30 uL of diluted compound from tube 11 add it to the tube 10 and mix gently by pipetting up and down Discard the pipet tip g Repeat this process 7 more times preparing serial dilutions from right to left across the plate DO NOT add modulator compound to tubes 1 and 2 These samples serve as the no modulator control and complete the dose curve h Repeat process when testing additional compounds i Set compou
11. d GPCR Internalization cells previously plated on day 1 from the incubator Transfer 5 uL from tubes 1 12 to each well according to the plate map on page 13 Incubate for 30 minutes at 37 C AGONIST COMPOUND PREPARATION AND ADDITION 1 During the antagonist incubation determine the ECgo concentration of the agonist from the agonist dose response curve described on pages 10 12 Prepare a 22X ECgo concentration of agonist compound as shown below Example If the ECgo of the agonist compound is 10 nM prepare a stock at 220 nM Add 5 uL of agonist compound to each well Add 5 uL of CP Reagent to the no agonist wells column 1 Incubate for 3 hours 25 C or 37 C NOTE Please refer to the cell line specific datasheet for any variation in assay conditions 14 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 pipet tip g Repeat this process 8 more times preparing serial dilutions from right to left across the tubes h DO NOT add agonist compound to tube 1 This sample serves as the no agonist control and completes the dose curve i Repeat this process for each compound to be tested j Set compounds aside until agonist compounds are ready to be added Remove PathHunter eXpress Activated GPCR Internalization cells previously plated on day 1 from the incubator Transfer 10 uL from tubes 1 12 to each well accor
12. ding to the plate map on page 10 Incubate for 3 hours 25 C or 37 C NOTE Please refer to the cell line specific datasheet for any variation in assay conditions SUBSTRATE PREPARATION AND ADDITION 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 Component Entire Plate 96 wells Cell Assay Buffer 4 75 mL Substrate Reagent 1 1 25 mL Substrate Reagent 2 0 25 mL NOTE The working solution is stable for up to 8 hours at room temperature Add 55 uL of prepared detection reagent per well and incubate for 60 minutes at room temperature 23 C DO NOT pipette up and down in the well to mix or vortex shake plates Read samples on any standard luminescence plate reader Use GraphPad Prism or other comparable program to plot your agonist dose response 11 QUICK START PROCEDURE AGONIST DOSE RESPONSE Plate 100 pL PathHunter eXpress cells well Incubate 24 or 48 hours 37 C Add 10 uL of Agonist Incubate 3 hours 25 C or 37 C Add 55 uL Detection Reagent Working Solution Incubate 60 Minutes Room Temperature Read Chemiluminescent Signal Please refer to the cell line specific datasheet for any variation in assay conditions 12 ASSAY PROCEDURE ANTAGONIST DOSE RESPONSE The steps outlined below provide t
13. en tration at 11 pM 13
14. endor source and catalog number can be found on the cell line specific datasheet For optimal assay performance we recommend using control ligands provided by DiscoveRx Visit www discoverx com ligands control_ ligands for the complete DiscoveRx offering 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 Confirm that the final ligand concentration is correct Some ligands are sticky and difficult to dissolve Confirm that the cell line responds to the control agonist 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 How long is the 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 user manual 20 PROTOCOL OV
15. he assay volumes and procedure for performing antagonist assays using the PathHunter eXpress Activated GPCR Internalization cells and PathHunter Detection Reagents Although plate layouts and experimental designs may vary we recommend performing an 11 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 p E 2 o oO z Ss S a 2 i ene 5 3 fold serial dilution of antagonist 2g 2 Low High 1 2 3 4 5 6 z 8 9 10 11 12 A Compound 1 B c Compound 2 D E F Compound 3 G Compound 4 H DAY 2 OR 3 ANTAGONIST COMPOUND PREPARATION AND ADDITION 1 Dissolve antagonist compound in the vehicle of choice DMSO Ethanol PBS or other at the desired concentration 2 Prepare 3 fold serial dilutions of antagonist compound in CP Reagent containing the appropriate solvent DMSO ethanol PBS or other The concentration of each dilution should be prepared at 22X of the final screening concentration i e 5 uL antagonist compound will be used in a final volume of 110 uL For each dilution the final concentration of solvent should remain constant Preparation of 11 point dose curve serial dilutions We recommend starting with a concentration that is 50X the expected ICso value for the compound 1100X ICs would be the final screening concentration Example If the expected ICs is 10 nM prepare the highest starting conc
16. list of commercially available luminometers 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 UL Analyst GT Gemini SpectraMax Flexstation LMax Tecan Ultra Evolution Beckman Coulter CRi Berthold Technologies Mithras LB 940 Hamamatsu FDSS6000 FDSS RayCatcher 21 FREQUENTLY ASKED QUESTIONS FO FO FO FO FO FO 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 line specific data sheet Can the source of my agonist or antagonist compound impact my assay performance Yes the vendor source of compound can impact assay performance dramatically 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 Each PathHunter eXpress target has been QC tested and validated using a reference ligand Information on the reference ligand used for each assay including the v
17. nds aside until they are ready to be added Remove PathHunter eXpress cells previously plated on day 1 from the incubator Transfer 5 uL from tubes 1 12 to each well according to the plate map on page 17 Incubate for 30 minutes 37 C AGONIST COMPOUND PREPARATION AND ADDITION 1 During the modulator compound incubation determine the ECio and ECoo concentration of the agonist from the agonist dose response curve described on pages 10 12 Prepare a 22X ECio concentration PAM or 22X ECoo concentration NAM of agonist compound as shown below Example If the ECio ECoo of the agonist compound is 10 nM prepare a stock at 220 nM Add 5 uL of agonist compound to each well Add 5 uL of CP reagent to the no agonist wells column 1 Incubate for 3 hours 25 C 37 C NOTE Please refer to the cell line specific datasheet for any variation in assay conditions SUBSTRATE PREPARATION AND ADDITION 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 Component Entire Plate 96 wells Cell Assay Buffer 4 75 mL Substrate Reagent 1 1 25 mL Substrate Reagent 2 0 25 mL NOTE The working solution is stable for up to 8 hours at room temperature Add 55 ul of prepared detection reagent per well and incubate for 60 minutes at room temperature 23 C DO NOT pipette up and
18. nt 1 and 1 part Substrate Reagent 2 Cell Assay Buffer 4 75 mL Substrate Reagent 1 1 25 mL Substrate Reagent 2 0 25 mL NOTE The working solution is stable for up to 8 hours at room temperature Add 55 uL of prepared detection reagent per well and incubate for 60 minutes at room temperature 23 C DO NOT pipette up and down in the well to mix or vortex shake plates Read samples on any standard luminescence plate reader Use GraphPad Prism or other comparable program to plot your antagonist dose response 15 a Label tubes 1 through 12 b Add 60 ul of CP Reagent to tubes 1 11 c Prepare a working stock of antagonist compound in the appropriate CP Reagent d Add 90 uL of the working concentration of antagonist compound to tube 12 e Remove 30 uL of diluted compound from tube 12 add it to tube 11 and mix gently by pipetting up and down Discard the pipet tip f With a clean pipet tip remove 30 uL of diluted compound from tube 11 add it to the tube 10 and mix gently by pipetting up and down Discard the pipet tip g Repeat this process 7 more times preparing serial dilutions from right to left across the plate h DO NOT add antagonist compound to tubes 1 and 2 These samples serve as the no antagonist controls and complete the dose curve i Repeat process when testing additional compounds j Set compounds aside until antagonist compounds are ready to be added Remove PathHunter eXpress Activate
19. ontaining appropri ate solvent For preparation of test compounds we recommend preparing the dilutions using the CP Reagent provided in the kit For antibodies or other com pounds that may be sensitive to serum 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 The kinetics of ligand induced receptor internalization can vary depending on the target and temperature used during the compound incubation step For optimal assay performance we recommend you perform the compound incubation step according to the protocol provided in the cell line specific datasheet Always use the incubation temperature recommended for the kit you are testing ASSAY PROCEDURE ALLOSTERIC MODULATOR DOSE RESPONSE The steps outlined below provide the assay volumes and procedure for performing allosteric modulator assays using the PathHunter eXpress Activated GPCR Internalization cells and PathHunter Detection Reagents Although plate layouts and experimental designs may vary we recommend performing an 11 point dose curve for each compound concentration using at least duplicate wells for each dilu tion The protocol and volumes described below are designed for a complete 96 well plate t pe 2 a 5 5 gt amp S oO A ra 5 2 2 3 fo E E 3 fold serial dilution of modulator S 2 L
20. ow High 1 3 4 5 6 7 8 9 10 11 12 Compound 1 Compound 2 Compound 3 ro7 FOO DB p Compound 4 DAY 2 OR 3 MODULATOR COMPOUND PREPARATION AND ADDITION 1 Dissolve modulator compound in the vehicle of choice DMSO Ethanol PBS or other at the desired concentration 2 Prepare 3 fold serial dilutions of modulator compound in CP Reagent containing the appropriate solvent DMSO ethanol PBS or other The concentration of each dilution should be prepared at 22X of the final screening concentration i e 5 yL modulator compound will be used in a final colume of 110 pL For each dilution the final concentration of solvent should remain constant Preparation of 11 point dose curve serial dilutions We recommend starting with a concentration that is 50X the expected ICso value for the compound 1100X ICs would be the final working concentration Example If the expected ICs is 10 nM prepare the highest starting concen tration at 11 uM This is the working concentration a Label tubes 1 through 12 b Add 60 uL of CP reagent containing appropriate solvent to tubes 1 11 c Prepare a working stock of modulator compound in the appropriate CP reagent d Add 90 ul of the working concentration of modulator compound to tube 12 17 QUICK START PROCEDURE ANTAGONIST DOSE RESPONSE Plate 100 uL PathHunter eXpress cells well Incubate 24 or 48 hours 37 C Add 5 uL of Antagonist
21. rnalization assays were developed using a single universal protocol that includes a 3 hour compound incubation step Is the EFC signal impacted by a change in pH No The enzyme fragments are always in the cytosol so the pH does not change gt O How much internalization is required before signal is detected The amount of internalization that occurs prior to ligand stimulation will definitely vary from target to target Activated Internalization assays are highly specific such that even if the receptor is internalizing in the absence of ligand you don t get signal because Arrestin is not recruited and EA is not present at the endosome with the receptor thus no complementation occurs gt O Can my eXpress assay be run in 384 well format All PathHunter eXpress assays are optimized and formatted to 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 PO For additional information or technical support please call 1 866 448 4864 NA or 44 121 260 6142 Europe or email us at info discoverx com 22 LEGAL SECTION This product and or its use is covered by one or more U S patents 7 135 325 B2 8 101 373 B2 and or foreign patent applications and trade secrets that are either owned by or licensed to DiscoveRx Corporation LIMITED US
22. tem the small 42 amino acid enzyme fragment of B gal called ProLink PK is localized to intracellular endosomes and the larger complementing enzyme fragment termed Enzyme Acceptor or EA is fused to B Arrestin Stimulation of the receptor results in Arrestin binding to the activated GPCR followed by internalization and trafficking of the receptor arrestin complex to cellular endosomes This action forces complementation of the two enzyme fragments resulting in the formation of a functional enzyme that is capable of hydrolyzing substrate and generating a chemilu minescent signal using PathHunter Detection Reagents G Si ti rate ubstrate Light Activated Figure 1 PathHunter eXpress Activated GPCR Internalization Assay Principle Activation of the GPCR results in internalization of the receptor to intracellular endosomes and formation of a functional p gal enzyme capable of hydrolyzing substrate and generating chemilu minescent signal 4 FREQUENTLY ASKED QUESTIONS CONTINUED gt O gt O gt O Why do longer incubation times with Detection Reagents lead to a higher signal The complemented f galactosidase f gal enzyme is continually turning over the substrate over time Theoretically the signal continues to increase until the substrate is depleted Therefore the longer you incubate the reaction the higher the RLU values What is the shelf life of the eXpress kits We recommend that eXpress kits should be
23. uired to use the cell line It is the purchaser s responsibility to de termine if such patents or other intellectual property rights are required INTENDED USE PathHunter eXpress Activated GPCR Internalization Assays are ready to use complete kits that contain everything you need to measure arrestin bound internalized GPCRs in live cells without the hassle of using antibodies radioactivity or elaborate imaging The eXpress kits include single use vials of frozen cells stably expressing the GPCR of interest optimized cell plating reagent 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 studying receptor recycling identifying functional antagonists or determining mechanism of action of your lead compounds the ready to assay eXpress format eliminates the need for lengthy expensive and time consuming cell culture and makes functional testing fast and convenient Assays are designed for 96 well plate analyses and kits include enough cells and detection reagents for either 100 200 or 1000 datapoints Test compounds are not included and must be provided by the researcher TECHNOLOGY PRINCIPLE PathHunter eXpress Activated GPCR Internalization Assays provide a quanti tative measurement of internalized GPCR protein localized to early endosome using B galactosidase B gal enzyme fragment complementation EFC Figure 1 In this sys
24. 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 specific expiration date information What if my Cell Plating Reagent changes from a red pink color to yellow after freezing thawing If the Cell Plating 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 will not affect assay performance Can I use this assay to test human plasma or serum samples Yes PathHunter eXpress Activated GPCR Internalization 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 CELL PLATING reagent before addition of the PathHunter Detection Reagents It has been shown that EDTA anti coagulated plasma inhibits EFC and should be avoided for these types of studies What instruments can I use to read the plates Any bench top luminometer will work with the PathHunter eXpress Activated GPCR Internalization Assays Below is a partial
25. ytoTracker LDH Quantification Kit DiscoveRx Cat 92 2002 Products not available in all countries Please inquire CytoTracker Glutathione Quantification Kit DiscoveRx Cat 92 2003 CytoTracker DNA Damage Quantification Kit DiscoveRx Cat 92 2004M ASSAY INCUBATION AND CELL PLATING REAGENT REQUIREMENTS Each PathHunter eXpress Activated GPCR Internalization assay has been validated for optimal assay performance at either 24 or 48 hours post thaw Although most targets perform similarly at both time points for optimal assay performance we recommend you perform the assay according to the protocol provided in the cell line specific datasheet using both the recommended time point and CP Reagent Always use the CP Reagent included in the kit and DO NOT substitute from an alternate kit at any time NOTE Use special caution when testing multiple targets in the same experiment as targets may have different incubation times and CP Reagent requirements COMPOUND PREPARATION AND INCUBATION TEMPERATURE PathHunter eXpress Activated GPCR Internalization Assays are routinely 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 To validate each PathHunter eXpress Activated GPCR Internalization Assay reference ligand was diluted in the recommended CP Reagent c
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