PathHunter® eXpress β-Arrestin Human and Ortholog GPCR Assays
Contents
1. QUICK START PROCEDURE ASSAY PROCEDURE ANTAGONIST DOSE RESPONSE PLATE MAP PROTOCOL QUICK START PROCEDURE ASSAY PROCEDURE ALLOSTERIC MODULATOR RESPONSE PLATE MAP PROTOCOL QUICK START PROCEDURE ASSAY PROCEDURE NEUTRALIZING ANTIBODY RESPONSE PLATE MAP PROTOCOL QUICK START PROCEDURE FREQUENTLY ASKED QUESTIONS 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 10 PAGE 12 PAGE 13 PAGE 13 PAGE 16 PAGE 17 PAGE 17 PAGE 19 PAGE 20 PAGE 20 PAGE 23 PAGE 24 APPENDIX A RELATED PRODUCTS Description Catalog Number For more information visit Control Ligands Many www discoverx com pathway_assays control_ligands php Cell Plating Reagents PathHunter eXpress B Arrestin GPCR Assays PathHunter eXpress B Arrestin Orphan GPCR Assays PathHunter eXpress B Arrestin Ortholog GPCR Assays 93 0563R Series Many Many Many 27 www discoverx com certified cell_plating_reagents www discoverx com gpcrs express_arrestin php www discoverx com gpcrs express_orphan php www discoverx com gpcrs express_ortholog php FREQUENTLY ASKED QUESTIONS CONTINUED Q gt Can I use the PathHunter eXpress f Arrestin GPCR Assays to study novel GPCR Heterodimers Yes To perform a heterodimer experiment transiently transfect the PathHunter eXpress cells2. Interaction assay Assay that employs genetically modified cells and vectors collectively the Cells and 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 p
3. Transfer 5 uL from tubes 1 12 to each well according to the plate map on p 17 5 Incubate for 30 minutes 37 C 2 AGONIST COMPOUND PREPARATION AND ADDITION 3 1 During the modulator compound incubation determine the EC o and ECoo concentration of the agonist from the agonist dose response curve described on pages 9 11 Prepare a 22X ECio concentration PAM or 22X EC o concen tration 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 2 Add 5 ul of agonist compound to each well Add 5 uL of CP reagent to the no agonist wells column 1 3 Incubate for 90 minutes 37 C 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 I 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 compounds aside until they are ready to be added NOTE Please refer to the cell line specific datasheet for any variation in assay conditions SUBSTRATE PREPARATION AND ADDITION 1 During the in
4. o noO High 1 2 3 4 5 6 7 8 9 10 11 12 Compound 1 Compound 2 Compound 3 zo7m g a EF Eki Bil ER ERE BEN BEN ENE EEE EW BEL NENA 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 working concentration Example If the expected ECs is 10 nM prepare the highest starting concen tration at 5 5 uM a 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 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 an
5. complete DiscoveRx offering GPCR Test compound s 96 well V bottom compound dilution plates DiscoveRx Cat 92 0011 Multi mode or luminescence plate reader SN SONE XURI sk Disposable Reagent Reservoir such as Thermo Scientific Cat 8094 or similar RECOMMENDED MATERIALS The following products are recommended e CytoTracker LDH Quantification Kit DiscoveRx Cat 92 2002 e CytoTracker Glutathione Quantification Kit DiscoveRx Cat 92 2003 e CytoTracker DNA Damage Quantification Kit DiscoveRx Cat 92 2004M Products not available in all countries Please inquire SOLVENTS AND PREPARATION OF COMPOUND DILUTIONS PathHunter eXpress B Arrestin 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 B Arrestin GPCR Assay reference ligand was diluted in CP reagent containing appropriate solvent For preparation of test com pounds we recommend preparing the dilutions using the CP reagent provided in the kit containing the appropriate solvent For antibodies or other compounds 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 withou
6. 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 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 26 LEGAL SECTION This product and or its use is covered by one or more of the following U S patents 6 342 345 B1 7 135 325 B2 8 101 373 B2 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
7. 7 C Add 10 uL of Agonist Incubate 90 minutes 37 C BO NM d Add 55 pL 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 ALLOSTERIC MODULATOR DOSE RESPONSE The steps outlined below provide the assay volumes and procedure for performing allosteric modulator assays using the PathHunter eXpress B Arrestin cells and De tection 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 dilution The protocol and volumes described below are designed for a complete 96 well plate 3 fold serial dilution of modulator Low High No modulator No agonist No modulator agonist RI N w A u a N J o 10 11 12 Compound 1 Hezi mani Ban kan Mazi Haw Had eni kam B man Ras Rawan Compound 3 La X7520414 4 J Ll J awewa za m mi g e FB gt 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
8. 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 DiscoverR 2014 DiscoveRx Corporation Fremont CA 94538 70 255 DRx_UM_PHexX_0914V6 All rights reserved DiscoveR PathHunter CA PRESS PathHunter eXpress Arrestin Human and Ortholog GPCR Assays User Manual Simple Solutions for Complex Biology CONTENTS LEGAL SECTION INTENDED USE TECHNOLOGY PRINCIPLE PROTOCOL OVERVIEW KIT CONTENTS AND STORAGE CONDITIONS RECOMMENDED MATERIALS MATERIALS PROVIDED ADDITIONAL MATERIALS REQUIRED NOT PROVIDED RECOMMENDED MATERIALS SOLVENTS AND PREPARATION OF COMPOUND DILUTIONS USE OF PLASMA OR SERUM CONTAINING SAMPLES ASSAY INCUBATION AND CELL PLATING REAGENT REQUIREMENTS THAWING AND PLATING FROZEN CELLS ASSAY PROCEDURE AGONIST DOSE RESPONSE PLATE MAP PROTOCOL
9. SE VIALS TO ROOM TEMPERATURE NOTE When removing cryovials from liquid N 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 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 Plate 100 uL of cells into each well of the provided 96 well tissue culture plate 7 After seeding the cells into the microplate incubate for either 24 or 48 hours at 37 C 5 CO gt 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 B Arrestin cells and Detection Reagents Although plate layouts and experimental designs may vary we recommend per forming 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 Ir E 9 3 3 fold serial dilution of agonist Low g
10. a Label tubes 1 through 12 b Add 60 ul of CP reagent containing appropriate solvent to tubes 1 11 13 4 5 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 DO NOT add antagonist compound to tubes 1 and 2 These samples serve as the no antagonist control and completes the dose curve h Repeat process when testing additional compounds i Set compounds 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 13 Incubate for 30 minutes 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 9 11 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
11. cubation 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 18 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 uL PathHunter express cells per well Incubate 24 or 48 hours 3
12. d 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 they are ready to be added Remove PathHunter express cells previously plated on day 1 from the incubator Transfer 10 uL from tubes 1 12 to each well according to the plate map on page 9 Incubate for 90 minutes 37 C NOTE Please refer to the cell line specific datasheet for any variation in assay conditions 10 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 per well Incubate 24 or 48 hours 37 C Add 5 uL of Allosteric Modulator Incubate 30 minutes Add 5 ul of Agonist Incubate 90 minutes 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 3 Remove PathHunter eXpress cells previously plated on day 1 from the incubator
13. e 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 d Add 90 ul of the working concentration of antibody to tube 12 e Remove 30 uL of diluted antibody 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 antibody 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 antibody to tubes 1 and 2 These samples serve as the no antibody control and complete the dose curve h Repeat process when testing additional antibodies i Set antibody dilutions aside until they are ready to be added 3 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 20 5 Incubate for 30 minutes 37 C AGONIST COMPOUND PREPARATION AND ADDITION 1 During the antibody incubation determine the ECgo concentration of the ago nist to be used in the assay Prepare a 22X ECs concentration of agonist compound as shown below Example If the expected ECgo of the agonist compound is 10 nM prepare a stock at 220
14. e working solution is stable for up to 8 hours at room temperature 2 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 3 Read samples on any standard luminescence plate reader Use GraphPad Prism or other comparable program to plot your antibody dose response 22 MATERIALS PROVIDED Description Contents Contents Contents Storage Box 1 1 vial 2 vials 10 vials 80 C short PathHunter eXpress 1 x 10 cells ea 1 x 10 cells ea 1 x 10 cells ea Liquid N2 B Arrestin Cells long Box 2 PathHunter Detection 100 dp 200 dp 1 000 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 2x 100 mL Box 3 1 plate 2 plates 10 plates Room 96 well Tissue Culture Temperature Treated Plates Centrifuge vial before opening to maximize recovery Refer to cell line specific data sheets for optimized Cell Plating Reagent included with each kit 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
15. ion of DMSO or Ethanol used for dilution is too high Maintain concentration of the agonist antagonist diluent at lt 1 A2 Confirm that the final ligand concentration is correct Some ligands are sticky and difficult to dissolve A3 Confirm that the cell line responds to the control agonist a Q My cells arrived thawed Can I use them A No Call technical support for a replacement Q How long is the prepared detection reagent good for A The working detection reagent solution must be used within 8 hours of mixing Q What instruments can I use to read the plates A Any bench top luminometer will work with the PathHunter eXpress Arrestin GPCR Assays Below is a partial 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 LIL Analyst HT LJL Analyst GT Gemini SpectraMax Flexstation LMax Tecan Ultra Evolution Beckman Coulter CRi Berthold Technologies Mithras LB 940 Hamamatsu FDSS6000 FDSS RayCatcher Q How long is the signal stable for A The signal is stable for 24 hours after addition of detection reagent Q My cells are floating after the 48 hours incubation A The ce
16. lls are not viable contact technical support for a replacement Q Can I switch plates or should I use the plate provided A You can use any clear bottom white or opaque walled plate Q What if cells are not completely adherent after 24 48 hrs incubation A 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 24 PROTOCOL OVERVIEW 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 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 GPCR activity using a PathHunter eXpress B Arrestin GPCR assay Figure 2 1 Thaw and plate frozen assay ready eXpress cells page 9 2 Dilute and add compounds or antibodies 3 Perform functional assay in agonist antagonist or allosteric modulator mode pages 9 13 and 17 Plate cells Incubate 24 or 48 hours at 37 C Add Compounds Incubate 90 minutes at 37 C Add PathHunter Detection Reagents Incubate 60 minutes at Room Temperature Read Chemiluminescence using a standard plate reader Figure 2 Monitor functional GPCR responses to compound challe
17. nM 2 Add 5 ul of agonist compound to each well Add 5 uL of CP reagent to the no agonist wells column 1 3 Incubate for 90 minutes 37 C 4 If samples do not contain plasma or serum omit step 5 and proceed directly to the Substrate Preparation and Addition section on page 22 NOTE Please refer to the cell line specific datasheet for any variation in assay conditions ATTENTION PLASMA OR SERUM CONTAINING SAMPLES ONLY 5 After incubation is complete gently aspirate the plasma or serum containing samples from the well Be careful to remove as much sample as possible without disturbing the cell monolayer 6 Immediately add 110 uL of fresh CP reagent to each well Proceed to the Substrate Preparation and Addition section on page 22 NOTE Additional CP reagent is required for this media exchange step Please visit www discoverx com certified cell_plating_reagents for ordering information 21 ASSAY PROCEDURE NEUTRALIZING ANTIBODY DOSE RESPONSE The steps outlined below provide the assay volumes and procedure for detection or anti GPCR neutralizing antibodies using the PathHunter eXpress B Arrestin cells and Detection Reagents Although plate layouts and experimental designs may vary we recommend performing a 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 3 fold serial dilutions of antibody L
18. 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 90 minutes 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 14 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
19. ng a reference ligand Information on the reference ligand used for each assay including the vendor 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 pathway_assays control_ligands php for the complete DiscoveRx offering What Protocol can I use for antibody studies You should use an antagonist protocol for detection of neutralizing antibodies Refer to pages 20 23 for more details Can I use this assay to test human plasma or serum samples Yes PathHunter eXpress B Arrestin 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 of the Path Hunter Detection Reagents It has been shown that EDTA anti coagulated plasma inhibits EFC and should be avoided for these types of studies 25 FREQUENTLY ASKED QUESTIONS Q I did not see a signal with my control agonist A 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 Q I did not see a response with my compound A1 The concentrat
20. nge using the fast and simple PathHunter eXpress protocol 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 CELLS 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 N2 storage use tongs and place immediately on dry ice in a covered container Wait at least one minute for any liquid N2 inside the vial to evaporate and proceed with the thawing protocol page 8 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 indica
21. o acid fragment of B gal called ProLink and co expressed in cells stably expressing a fusion protein 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 to the ProLink tagged GPCR and forces 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 C with ProLink Stimulation amp Translocation p Arrestin EA o Oe rel Substrate o B Gal EA Acceptor Light B Arrestin EA Inactive beta gal Figure 1 PathHunter B 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 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 galactosidase gal enzyme is continually turning over the substrate Theoretically the signal continues to increase until the substrate is depleted Therefore the longer you incubate the reacti
22. 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 ICs value for the compound 1100X ICs would be the final working concentration Example If the expected ICso 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 L 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 pL PathHunter eXpress cells per well Incubate 24 or 48 hours 37 C Add 5 uL of Antagonist Incubate 30 minutes 37 C Add 5 uL of Agonist ECgo Incubate 90 minutes 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 16 ASSAY PROCEDURE ANTAGONIST DOSE RESPONSE The steps outlined below provide the as
23. on the higher the RLU values What is 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 specific 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 f Arrestin GPCR assay to test inverse agonist compounds Yes For compounds that behave as inverse agonists follow the agonist protocol as described on page 10 Can the source of my agonist or antagonist 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 Each PathHunter eXpress target has been QC tested and validated usi
24. ow lt High 3 4 5 6 7 8 9 10 1i 12 I No antibody No agonist IN No antibody agonist Compound 1 Compound 2 Compound 3 Compound 4 T O Tw m o ow gt DAY 2 OR 3 ANTIBODY PREPARATION AND ADDITION Dissolve antibody in the vehicle of choice at the desired concentration 2 Prepare 3 fold serial dilutions of antibody in CP reagent containing the appropriate solvent The concentration of each dilution should be prepared at 22X of the final screening concentration i e 5 uL antibody 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 ICs value for the antibody 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 a Label tubes 1 through 12 b Add 60 L of CP reagent containing appropriate solvent to tubes 1 11 c Prepare a working stock of antibody in CP Reagent containing appropriate solvent 20 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 CP reagent in a 37 C water bath 2 Remove cell vial s from 80 C or liquid N vapor phase storage and place immediately on dry ice prior to thawing DO NOT EXPO
25. roducts cell lines certain 3 party gene specific patents may be required 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 B Arrestin Human and Ortholog 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 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 measuring one or multiple GPCR responses to compound challenge 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 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 f gal enzyme fragment complementation EFC Figure 1 In this system the GPCR of interest is fused in frame with the small 42 amin
26. say volumes and procedure for performing antagonist assays using the PathHunter eXpress B Arrestin cells and 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 Pe u i 2 9 E 5 o z 23 8 c r a 9 2 3 fold serial dilution of antagonist 2 2 Low High 1 2 3 4 5 6 7 8 9 10 11 12 Compound 1 Compound 2 Compound 3 CEC a a 1 L PP 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 working concentration Example If the expected ICs is 10 nM prepare the highest starting concen tration at 11 uM
27. t affecting assay performance Some assays require compound incubation times and temperatures that differ from the protocol described in this user manual For optimal assay performance we recommend you perform the assay according to the protocol provided in the cell line specific datasheet USE OF PLASMA OR SERUM CONTAINING SAMPLES PathHunter eXpress B Arrestin 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 i e 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 Refer to page 20 for more information 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 ASSAY INCUBATION AND CELL PLATING REAGENT REQUIREMENTS Each PathHunter eXpress B Arrestin Human and Ortholog GPCR 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 th
28. tes 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 working solution is stable for 24 hours at room temperature Box 3 96 WELL TISSUE CULTURE TREATED PLATES Store at Room Temperature QUICK START PROCEDURE NEUTRALIZING ANTIBODY RESPONSE Plate 100 uL PathHunter express cells per well Incubate 24 or 48 hours 37 C Pe wus Add 5 uL of Diluted Antibody Incubate 30 minutes 37 C Add 5 uL of Agonist ECgo Incubate 90 minutes 37 C FOR SERUM RN RE SAMPLES ONLY Remove serum containing sample Add 110 uL CP Reagent 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 23 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 Substrate Reagent 1 Substrate Reagent 2 NOTE Th
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