PathHunter® β-Arrestin GPCR Assays
Contents
1. Compound 5 Compound 6 Compound 7 Compound 8 Compound 10 Compound 9 Compound 11 Compound 12 Compound 13 Compound 14 O Compound 15 Compound 16 Figure 3 This plate map shows 12 point dose curves with 2 data points at each concentration Plate map allows 16 compounds to be tested in duplicate per 384 well plate DAY 1 PREPARATION OF ASSAY PLATES Plate PathHunter cells in the appropriate number of wells in a 384 well plate as described in the Preparation of Assay Plates section on p 9 Allow cells to incu bate overnight 10 DAY 2 AGONIST COMPOUND PREPARATION AND ADDITION L Dissolve agonist compound in the vehicle of choice DMSO ethanol PBS or other at the desired stock concentration Prepare a series of twelve 3 fold serial dilutions of agonist compound in CP Reagent containing the appropriate solvent DMSO ethanol PBS or other as described below The concentration of each dilution should be prepared at 5X of the final screening concentration i e 5 uL compound 20 uL of cells For each dilution the final concentration of solvent should remain constant To prepare the 12 point dose curve serial dilutions we recommend starting with a concentration that is 50X the expected ECs value for the compound e g 250X EC5o would be the final working concentration Example If the expected ECs is 10 nM prepare the highest starting concen tration of the corresponding dilution at 2 5 uM Th2. antagonist incubation is complete add 2 5 uL of agonist compound to wells 2 12 Add 2 5 uL of CP Reagent containing appropriate solvent to the No antagonist No agonist wells columns 1 amp 13 in Figure 5 Incubate for 90 minutes 37 C 15 SUBSTRATE PREPARATION AND ADDITION 1 Prepare PathHunter Detection Reagent by combining 1 part Substrate Reagent 2 Substrate with 5 parts Substrate Reagent 1 and 19 parts of PathHunter Cell Assay Buffer Entire Plate 384 wells 4 75 mL 1 25 mL 0 25 mL NOTE The working solution is stable for up to 8 hours at room temperature 2 Add 12 uL of prepared detection reagent to the appropriate wells DO NOT pipet up and down in the well to mix or vortex shake plates 3 Incubate for 60 minutes at room temperature 23 C Read samples on any standard luminescence plate reader Use GraphPad Prism or other comparable program to plot your antagonist dose response REPRESENTATIVE DATA AND DATA ANALYSIS Propanolol Timolol CGP12177a Carvedilol Atenolol Nadolol Bee lt gt p gt E 10 1110 10 10 2 10 8 10 7 10 10 5 10 4 10 3 Antagonist M 500 nM Isoproterenol Figure 6 PathHunter CHO K1 ADRB2 B Arrestin Cells 93 0182C2 Cells were plated in a 384 well plate at 5 000 cells well and levels of B Arrestin recruitment was measured after 30 minutes of pre incubation with the indicated concentrations of antagonist compounds followed by a 90 minute incubation wi
3. concentration Example If the expected ICs is 10 nM prepare the highest starting concen tration of the corresponding dilution at 5 uM This is the working concentration a For each antibody tested label the wells of a 384 well dilution plate 1 through 12 Add 20 uL of CP Reagent containing appropriate solvent to wells 1 11 Prepare a working concentration of antibody in the appropriate CP Reagent Add 30 uL of the working concentration of antibody to well 12 Remove 10 uL of antibody from well 12 add it to well 11 and mix gently by pipetting up and down Discard pipet tip f With a clean pipet tip remove 10 uL of diluted antibody from well 11 add it to well 10 and mix gently by pipetting up and down Discard the pipet tip g Repeat this process 7 more times in succession to prepare serial dilutions for the remaining wells from right to left across the plate DO NOT add antibody to wells 1 and 2 This sample serves as the no antibody control and completes the dose curve h Repeat this process for each additional antibody to be tested i Set antibodies aside until they are ready to be added mano Remove PathHunter cells from the incubator previously plated on day 1 Transfer 2 5 uL from wells 1 12 to duplicate wells according to the plate map shown on p 22 Incubate for 30 minutes 37 C AGONIST COMPOUND PREPARATION AND ADDITION 1 During the antibody incubation determine the ECg concentratio
4. of Revive Media to each T225 flask Rinse the cells from the surface of the flask using the added media Remove the cells from the flask and transfer to a 50 mL conical tube If necessary add an additional 5 mL of media to the flask and rinse to collect the remaining cells and transfer the additional volume to the 50 mL conical tube Remove 0 5 mL of the resus pended cells and count the cells using a hemocytometer Centrifuge the collected cells at 300 x g for 4 minutes After centrifugation discard the supernatant Resuspend the cell pellet in Preserve Freezing Reagent Based on the cell number obtained from Step 5 dilute the resuspended cells to a concentration of 1 2 x 10 cells mL using Preserve Freezing Reagent Transfer 1 mL cells to each 2 mL cryogenic tube Keep cells on ice during this process and transfer to a cryogenic container pre chilled at 4 C Transfer tubes to 80 C and store overnight Transfer tubes into the vapor phase of a liquid N tank for long term storage PREPARATION OF ASSAY PLATES Each PathHunter 8 Arrestin GPCR Assay has been validated for optimal assay per formance using the specific PathHunter Cell Plating Reagent Always use the CP Reagent recommended for the cell line and DO NOT substitute at any time de Harvest the cells as follows from a confluent T25 or T75 flask using Cell Detach ment Reagent Do not use Trypsin a Remove PathHunter select Cell Culture Media b Gently was
5. of antibody High PSS a en ae i 1 2 3 9 10 11 12 13 Antibody 4 A w Antibody 2 B Antibody 3 Antibody 4 Antibody 5 HHH 4 1 Antiboay 6 Antibody 7 3 H Antibody 8 Antibody 9 A FF ia k TTT antitoay 10 bed Antibody 11 Antibody 12 Antibody 13 Ee 1 Antibody 14 Antibody 15 Antibody 16 Figure 8 This plate map shows a 11 point dose curves with 2 data points at each concentration Plate layout allows 16 antibodies to be tested in duplicate per 384 well plate DAY 1 PREPARATION OF ASSAY PLATES Plate PathHunter cells in the appropriate number of wells in a 384 well plate as described in the Preparation of Assay Plates section on p 9 Allow cells to incu bate overnight DAY 2 ANTIBODY PREPARATION AND ADDITION 1 Dissolve antibody in the vehicle of choice PBS water or other at the desired stock concentration 2 Prepare a series of eleven 3 fold serial dilutions of antibody in Cell Plating Reagent containing the appropriate solvent PBS water or other The concentration of each dilution should be prepared at 10X of the final screening concentration i e 2 5 uL antibody will be used in a final volume of 25 uL For each dilution the final concentration of solvent should remain constant 22 To prepare the 11 point dose curve serial dilutions we recommend starting with a concentration that is 50X the expected ICs value for the compound e g 500X ICs would be the final working
6. DiscoveR PathHunter p Arrestin GPCR Assays For Chemiluminescent Detection of Activated GPCRs User Manual Simple Solutions for Complex Biology CONTENTS LEGAL SECTION INTENDED USE TECHNOLOGY PRINCIPLE ASSAY OVERVIEW MATERIALS PROVIDED ADDITIONAL MATERIALS REQUIRED FROZEN CELL HANDLING PROCEDURE CELL PLATING REAGENT REQUIREMENTS SOLVENTS AND PREPARATION OF COMPOUND DILUTIONS USE OF PLASMA OR SERUM CONTAINING SAMPLES STORING amp REMOVING CRYOVIALS FROM LIQUID NITROGEN CELL THAWING AND PROPAGATION CELL FREEZING PROTOCOL PREPARATION OF ASSAY PLATES ASSAY PROCEDURE AGONIST DOSE RESPONSE PROTOCOL QUICK START PROCEDURE ASSAY PROCEDURE ANTAGONIST DOSE RESPONSE PROTOCOL QUICK START PROCEDURE ASSAY PROCEDURE ALLOSTERIC MODULATOR RESPONSE PROTOCOL QUICK START PROCEDURE ASSAY PROCEDURE NEUTRALIZING ANTIBODY RESPONSE PROTOCOL QUICK START PROCEDURE TROUBLESHOOTING GUIDE APPENDIX A ASSAY FORMATS APPENDIX B RELATED PRODUCTS PAGE 3 PAGE 4 PAGE 4 PAGE 5 PAGE 5 PAGE 6 PAGE 6 PAGE 6 PAGE 7 PAGE 7 PAGE 7 PAGE 8 PAGE 9 PAGE 9 PAGE 10 PAGE 13 PAGE 14 PAGE 17 PAGE 18 PAGE 21 PAGE 22 PAGE 25 PAGE 26 PAGE 28 PAGE 28 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 a
7. allows 16 compounds to be tested in duplicate per 384 well plate DAY 1 PREPARATION OF ASSAY PLATES Plate PathHunter cells in the appropriate number of wells in a 384 well plate as described in the Preparation of Assay Plates section on p 9 Allow cells to incu bate overnight DAY 2 ANTAGONIST COMPOUND PREPARATION AND ADDITION 1 Dissolve your antagonist compound in the vehicle of choice DMSO ethanol PBS or other at the desired stock concentration 2 Prepare a series of eleven 3 fold serial dilutions of antagonist compound in CP Reagent containing the appropriate solvent DMSO ethanol PBS or other as described below The concentration of each dilution should be prepared at 10X the final screening concentration i e 2 5 uL antagonist compound will be used in a final volume of 25 uL For each dilution the final concentration of 14 solvent should remain constant To prepare the 11 point dose curve serial dilutions we recommend starting with a concentration that is 50X the expected ICso value for the compound e g 500X ICs would be the final working concen tration Example If the expected ICs is 10 nM prepare the highest starting concen tration of the corresponding dilution at 5 uM This is the working concentration a For each compound tested label the wells of a 384 well dilution plate 1 through 12 Add 20 uL of CP Reagent containing appropriate solvent to wells 1 11 c Prepare a working concent
8. ap on p 18 Incubate cells with modulator compounds for 30 minutes 37 C AGONIST COMPOUND PREPARATION AND ADDITION 1 During the modulator compound incubation determine the EC1o EC9o concen tration of the agonist from the agonist dose response curve described on p 10 13 Prepare a 10X ECio concentration PAM or 10X ECo concentration NAM of agonist compound in the appropriate CP Reagent solvent as shown below Example If the expected EC1o ECo of the agonist compound is 10 nM prepare a stock at 100 nM When the modulator incubation is complete add 2 5 uL of agonist compound to well 2 12 Add 2 5 uL of CP Reagent containing appropriate solvent to the No modulator No agonist wells columns 1 amp 13 in Figure 7 Incubate for 90 minutes 37 C 19 SUBSTRATE PREPARATION AND ADDITION 1 Prepare PathHunter Detection Reagent by combining 1 part Substrate Reagent 2 Substrate with 5 parts Substrate Reagnet 1 and 19 parts of PathHunter Cell Assay Buffer Component Entire Plate 384 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 2 Add 12 uL of prepared detection reagent to the appropriate wells 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 Incubate for 60 minutes at room temperature 23 C Read sa
9. athHunter cells are stable reduced performance up to 10 passages Use low passage cells whenever possible Cells are not adherent and Confirm adherence of cells exhibit incorrect using microscopy morphology Low or No Signal Improper preparation of Detection reagents should detection reagents be prepared just prior to use and are sensitive to light Problem with cell growth See datasheet for cell cell viability cell adherence culture conditions or cell density Problem with microplate Microplate reader should be reader in luminescence mode Read at 1 sec well Experimental S B does For cell pools S B may vary Prepare a clonal cell line or not match datasheet greatly from passage to use lower passage number value passage or day to day cells Repeat the assay Confirm assay conditions Improper preparation of Some ligands are difficult ligand agonist or antagonist to handle Confirm the final concentration of ligands 26 TROUBLESHOOTING GUIDE CONTINUED PROBLEM CAUSE SOLUTION Cells growing slowly ECso is rig ht shifted High well to well vari ability in Z study U20S grows slower than CHO K1 or HEK 293 Slow growing clones Improper ligand handling or storage Difference in agonist binding affinity Problems with plate type and compound stability Problems with plate type and compound solubility Average doubling time is 3 days so please observe cells under mic
10. coveRx 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 Dis coveRx 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 DiscoveRx Corporation Attn Licensing Department 42501 Albrae Street Fremont CA 94538 tel 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 purchaser s responsibility to determine if such patents or other intellectual property rights are required INTENDED USE PathHunter B Arrestin GPCR Assays are whole cell functional assays that directly measure GPCR activity by detecting the interaction of B Arrestin with the activated GPCR Because Arrestin recruitment occurs independent of G protein coupling PathHunter B Arrestin assays offer a powerful and universal screening platform that can be used with virtually any Gi Gs or Gq coupled receptor This PathHunter system combines engineered clonal cell lines stably expressing the ProLink PK tagged GPCR of interest and the Enzyme acceptor EA tagged B Arrestin fus
11. ct assay performance optimize the assay conditions accordingly if other solvents or solvent concentrations are required To validate each PathHunter B Arrestin GPCR Assay reference ligand was diluted using the Cell Plating CP Reagent recommended for the cell line 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 without affecting assay performance USE OF PLASMA OR SERUM CONTAINING SAMPLES PathHunter 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 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 Refer to p 22 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 STORING amp REMOVING CRYOVIALS FROM LIQUID NITROGEN Cells are shipped in 2 vials on dry ice and contain approximately 1 x 10 cells per vial in 1 mL of Preserve Freezing Reagent The fo
12. e cell pellet is almost completely thawed Caution Longer incubation may result in cell death 3 To remove DMSO from the media carefully transfer the thawed cells to a sterile 15 mL tube and then fill tube with 10 mL pre warmed Revive Media Centri fuge at 300 x g for 4 minutes to pellet cells 4 Remove media without disturbing cell pellet and resuspend in 5 mL of pre warmed Revive Media Transfer cells to a T25 flask and incubate for 24 hours at 37 C 5 CQO NOTE Cell recovery is greatly improved when selection antibiotics are omitted for the first 24 hours 5 After 24 hours gently remove Revive Media being careful not to disturb the cell monolayer and replace with 5 mL of pre warmed complete PathHunter select Cell Culture Media 6 Once the cells become gt 70 confluent in the T25 flask aspirate media and wash cells with 5 mL PBS Aspirate PBS and dissociate cells with 0 5 mL Cell Detachment Reagent and resuspend in 5 mL of complete PathHunter select Cell Culture Media Transfer the entire cell suspension to a T75 flask containing 15 mL of PathHunter select Cell Culture Media for continued growth 7 Passage the cells every 2 3 days based on the doubling time of the cell line using cell Detachment Reagent For routine passaging prepare a 1 3 dilution of cells in a total volume of 15 mL PathHunter select Cell Culture Media Transfer 5 mL of the diluted cells to each new T75 flask NOTE To maintain logarith
13. h cells with 5 mL PBS and aspirate c Add 0 5 mL Cell Detachment Reagent to each T25 flask or 1 mL to each T75 flask d Place the flask in the incubator for 5 minutes or until cells have detached e Add 3 mL of CP Reagent and transfer to a 15 mL conical tube 9 2 Determine the cell density using a hemocytometer Centrifuge the cells at 300 x g for 4 minutes to pellet cells Remove supernatant 3 Resuspend cells in CP Reagent at a concentration of 250 000 cells mL 5 000 cells 20 uL Transfer 20 uL of the cell suspension to each well of a 384 well microplate Please refer to Appendix A for cell numbers and volumes for alternate formats 4 Incubate the plate overnight at 37 C 5 CO3 ASSAY PROCEDURE AGONIST DOSE RESPONSE CURVE The steps outlined below provide the assay volumes and procedures for performing GPCR agonist assays using the PathHunter B Arrestin GPCR Cell Lines and PathHunter Detection Reagents in a 384 well format Refer to Appendix A for cell numbers and volumes for alternate formats 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 w xa c O O D 3 fold serial 3 fold serial S Low dilutions of agonist High S Low dilutions of agonist High A E A E S 1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Compound 1 Compound 2 Compound 3 Compound 4
14. ion proteins with optimized PathHunter Detection Reagents Cat 93 0001 93 0001L and 93 0001XL Each cell line has been characterized for appropriate GPCR phar macology specificity and stability in cell culture By combining a simple one step addition protocol and standard chemiluminescent detection these assays are ideally suited for 96 well 384 well or 1536 well compound screening TECHNOLOGY PRINCIPLE PathHunter B Arrestin cell lines monitor GPCR activity by detecting the interaction of B Arrestin with the activated GPCR using B galactosidase B gal 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 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 at Translo ca tron GPCR Tagged with ProLink Pk E P gt 0050 gt Sub
15. is is the working concentration a For each compound tested label the wells of a 384 well dilution plate 1 through 12 b Add 20 uL of CP Reagent containing appropriate solvent to wells 1 11 Prepare a working concentration of agonist compound in the appropriate CP Reagent Add 30 uL of the working concentration of agonist compound to well 12 Remove 10 uL of compound from well 12 add it to well 11 and mix gently by pipetting up and down Discard pipet tip f With a clean pipet tip remove 10 uL of diluted compound from well 11 add it to well 10 and mix gently by pipetting up and down Discard the pipet tip g Repeat this process 8 more times in succession to prepare serial dilutions for the remaining wells from right to left across the plate DO NOT add agonist compound to well 1 This sample serves as the no agonist control and completes the dose curve h Repeat this process for each additional agonist compound to be tested i Set compounds aside until agonist compounds are ready to be added Remove PathHunter cells from the incubator previously plated on day 1 NOTE 93 0203C7 PathHunter C2C12 CXCR4 Arrestin cell line uses an additional media exchange step Please refer to cell line specific datasheet Transfer 5 uL from wells 1 12 to duplicate wells according to the plate map shown on p 10 Incubate for 90 minutes 37 C 11 SUBSTRATE PREPARATION AND ADDITION 1 Prepare PathHunter Detection Reagen
16. llowing procedures are for safely storing and removing cryovials from liquid nitrogen storage 1 PathHunter cells must arrive in a frozen state on dry ice If cells arrive thawed do not proceed contact technical support 2 Frozen cells must be immediately transferred to liquid Nz storage or thawed and put into culture upon arrival 3 When removing cryovials from liquid Nz 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 4 Proceed with the thawing protocol in the following section SAFETY WARNING A face shield gloves and lab coat should be worn at all times when handling frozen vials Some cryovials can leak when submerged in liquid N2 Upon thawing the liquid N present in the cryovial converts back to its gas phase which can result in the vessel exploding CELL THAWING AND PROPAGATION The following procedures are for thawing seeding and expanding the cells and for maintaining the cultures once the cells have been expanded Cells are free of contamination prior to shipment and care should be taken in their handling to avoid contamination NOTE Face shield gloves and a lab coat should be worn during the thawing procedure Pre warm 15 mL Revive Media in a 37 C water bath 2 Place the frozen cell vials briefly 10 seconds to 1 min in a 37 C water bath under sterile conditions until only small ice crystals remain and th
17. mic growth of the cells cultures should be maintained in a subconfluent monolayer 8 Each PathHunter B Arrestin GPCR Cell Line has been found to be stable for at least 10 passages with no significant drop in assay window or shift in ECso 9 Assay performance and cellular response can be assessed by treating the cells with reference agonist Refer to the cell line specific datasheet for the recommended control agonist for your PathHunter B Arrestin GPCR Cell Line For antagonist assays cells can be pretreated with varying doses of antagonist inhibitor compounds followed by agonist challenge typically at an ECgo concentration CELL FREEZING PROTOCOL The following procedures are for freezing cells from confluent T225 flasks If smaller flasks are used adjust the volumes accordingly Care should be taken in handling to avoid contamination l Remove T225 flasks from incubator and place in the tissue culture hood Aspirate the media from the flasks Add 10 mL PBS into each T225 flask and swirl to rinse the cells Aspirate PBS from flask Add 5 mL of Cell Detachment Reagent to the flask Rock the flask back and forth gently to ensure the surface of the flask is covered Incubate at 37 C 5 CO for 2 5 minutes or until the cells have detached Remove the flask from the incubator and view under a microscope to confirm that the cells have detached If necessary tap the edge of the flask to detach cells from the surface Add 8 10 mL
18. mples on any standard luminescence plate reader Use GraphPad Prism or other comparable program to plot your allosteric modulator dose response 20 QUICK START PROCEDURE ALLOSTERIC MODULATOR DOSE RESPONSE Plate 20 uL PathHunter cells well Incubate overnight 37 C Add 2 5 uL of Allosteric Modulator Compound Incubate 30 minutes 37 C Add 2 5 uL of Agonist Incubate 90 minutes 37 C Add 12 uL Detection Reagent Working Solution Incubate 60 Minutes Room Temperature Read Chemiluminescent Signal Please refer to the cell line specific datasheet for any variations in assay conditions 21 ASSAY PROCEDURE NEUTRALIZING ANTIBODY DOSE RESPONSE The steps outlined below provide the assay volumes and procedure for performing detection of anti GPCR neutralizing antibodies using the PathHunter B Arrestin GPCR Cell Lines and PathHunter Detection Reagents in a 384 well format Refer to Appendix A for cell numbers and volumes for alternate formats 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 384 well plate i g S lt gs amp OS oe x Sa Fa E z ar og gt 3 oe e l 3 fold serial E e 3 fold serial L Low dilutions of antibody High we we Low dilutions
19. n of the agonist to be used in the assay Prepare a 10X ECgo concentration of agonist compound as shown below Example If the expected ECgo of the agonist compound is 10 nM prepare a stock at 100 nM Add 2 5 uL of agonist to each well Add 2 5 uL of CP Reagent containing appro priate solvent to the no agonist wells columns 1 amp 13 in figure 8 Incubate for 90 minutes 37 C If samples do not contain plasma or serum omit steps 5 and 6 and proceed directly to the substrate preparation and addition 23 ATTENTION PLASMA OR SERUM CONTAINING SAMPLES ONLY 3 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 Immediately add 25 uL of fresh CP Reagent to each well Proceed with substrate preparation and addition SUBSTRATE PREPARATION AND ADDITION f Prepare PathHunter Detection Reagent by combining 1 part Substrate Reagent 2 Substrate with 5 parts Substrate Reagent 1 and 19 parts of PathHunter Cell Assay Buffer Entire Plate 384 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 12 uL of prepared detection reagent to the appropriate wells and incubate for 60 minutes at room temperature 23 C DO NOT pipette up and down in the well to mix or vortex shake
20. ntration that is 50X the expected ICs value 18 for the compound e g 500X ICs would be the final working concentration Example If the expected ICs is 10 nM prepare the highest starting concen tration of the corresponding dilution at 5 uM This is the working concentration a For each compound tested label the wells of a 384 well dilution plate 1 through 12 Add 20 uL of CP Reagent containing appropriate solvent to wells 1 11 Prepare a working concentration of modulator compound in the appropriate CP Reagent d Add 30 uL of the working concentration of modulator compound to well 12 e Remove 10 uL of compound from well 12 add it to well 11 and mix gently by pipetting up and down Discard the pipet tip f With a clean pipet tip remove 10 uL of diluted compound from well 11 add it to well 10 and mix gently by pipetting up and down Discard the pipet tip g Repeat this process 7 more times in succession to prepare serial dilutions for the remaining wells or from right to left across the plate DO NOT add modulator compound to wells 1 and 2 These samples serve as the no modulator controls and complete the dose curve h Repeat this process for any additional modulator compounds to be tested i Set compounds aside until you are ready to add them to the cells Remove PathHunter cells from the incubator previously plated on day 1 Transfer 2 5 uL from wells 1 12 to duplicate wells according to the plate m
21. plates Incubate for 60 minutes at room temperature 23 C Read samples on any standard luminescence plate reader Use GraphPad Prism or other comparable program to plot your dose response 24 QUICK START PROCEDURE NEUTRALIZING ANTIBODY RESPONSE Plate 20 uL PathHunter cells well Incubate overnight 37 C Add 2 5 uL of Diluted Antibody Incubate 30 minutes 37 C Add 2 5 uL of Agonist ECgo Incubate 90 minutes 37 C FOR SERUM SAMPLES ONLY Remove serum containing sample Add 25 uL CP Reagent Add 12 uL Detection Reagent Working Solution Incubate 60 Minutes Room Temperature Read Chemiluminescent Signal Please refer to the cell line specific datasheet any variations in assay conditions 25 TROUBLESHOOTING GUIDE PROBLEM CAUSE SOLUTION No Response Improper cell growth See datasheet for cell conditions culture conditions High DMSO solvent Maintain DMSO solvent at concentration lt 1 in serial dilutions of compounds Improper ligand used or See datasheet for recom improper ligand incubation mended ligand and assay time conditions Improper preparation of Refer to vendor specific ligand agonist or datasheet to ensure proper antagonist handling dilution and storage of ligand Improper time course for Optimize time course of induction induction with agonist and antagonist Decreased Response Higher passages give P
22. r monitoring GPCR activity in response to compound challenge MATERIALS PROVIDED PathHunter B Arrestin GPCR Cells Liquid N vapor phase Please refer to the cell line specific datasheet for detailed information on the PathHunter 8 Arrestin cell line you are testing ADDITIONAL MATERIALS REQUIRED The following additional materials are required to perform PathHunter B Arrestin GPCR Assays Green V Bottom PP Ligand Dilution Plates 10 plates pack DiscoveRx Cat 92 0011 96 well Clear Bottom TC treated Sterile WCB FB w lid 10 plates pack DiscoveRx Cat 92 0014 384 well Clear Bottom TC treated Sterile WCB FB w lid 10 plates pack DiscoveRx Cat 92 0013 384 well White Bottom TC treated Sterile w lid 10 plates pack DiscoveRx Cat 92 0015 Disposable Reagent Reservoir Thermo Scientific Cat 8094 or similar Hemocytometer Cryogenic Freezing Container Nalgene Cat 5100 0001 or similar PathHunter Detection Kit DiscoveRx Cat 93 0001 93 0001L or 93 0001XL Revive Media DiscoveRx Cat 92 0016RM Series PathHunter select Cell Culture Kits DiscoveRx Cat 92 0018G Series Preserve Freezing Reagent DiscoveRx Cat 92 0017FR Series Cell Detachment Reagent DiscoveRx Cat 92 0009 PathHunter Cell Plating CP Reagent DiscoveRx Cat 93 0563R Series Phosphate buffered saline PBS GPCR control agonist GPCR test compound s and or antagonists Cr
23. ration of antagonist compound in the appropriate CP Reagent d Add 30 uL of the working concentration of antagonist compound to well 12 Remove 10 uL of compound from well 12 add it to well 11 and mix gently by pipetting up and down Discard the pipet tip f With a clean pipet tip remove 10 uL of diluted compound from well 11 add it to well 10 and mix gently by pipetting up and down Discard the pipet tip g Repeat this process 7 more times in succession to prepare serial dilutions for the remaining wells or from right to left across the plate DO NOT add antagonist compound to tubes 1 and 2 These samples serve as the no antagonist controls and complete the dose curve h Repeat process for any additional antagonist compounds to be tested i Set compounds aside until you are ready to add them to the cells Remove PathHunter cells from the incubator previously plated on day 1 Transfer 2 5 uL from wells 1 12 to duplicate wells according to the plate map on p 14 Incubate cells with antagonist compounds for 30 minutes 37 C AGONIST COMPOUND PREPARATION AND ADDITION is During the antagonist incubation determine the ECgo concentration of the agonist from the agonist dose response curve described on p 10 13 Prepare a 10X ECgo concentration of agonist compound in the appropriate CP Reagent solvent as shown below Example If the expected ECg of the agonist compound is 10 nM prepare a stock at 100 nM When the
24. re either owned by or licensed to DiscoveRx Corporation This product is for in vitro use only and in no event can this product be used in whole animals The right to use or practice the inventions in the foregoing patents including method of use claims by using or propagating this product is granted solely in connection with the use of appropriate Detection Reagents protected un der trade secret purchased from DiscoveRx Corporation or its authorized distributors 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 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 Dis
25. roscope and monitor cell health Use of DiscoveRx functionally validated and optimized media and reagents improves assay performance Check ligand handling requirements Confirm that the ligand used is comparable to the ligand in the Product Insert Hydrophobic compounds should be tested for solubility and may be diluted in buffer containing 0 1 BSA Non binding surface plates may be necessary for hydrophobic compounds Z studies should be performed with automation It may be necessary to test plate types and compound stability For additional information or technical support please call 1 866 448 4864 US 44 121 260 6142 Europe or email info discoverx com 27 APPENDIX A ASSAY FORMATS PathHunter Certified Assay Format Plate Format 96 well FV 384 well LV 384 well 1536 well rcaumamers ioo s00 2500 Cell Plating Reagent volume used to resuspend cells for assay plates APPENDIX B RELATED PRODUCTS Control Ligands www discoverx com pathway_assays control_ligands php PathHunter Cell Plating Reagents www discoverx com certified cell_plating_reagents php PathHunter Certified Cell Culture www discoverx com certified PH_cell Reagents culture_reagents php e PathHunter select Cell Culture www discoverx com certified PH_cell Kit culture_reagents php e Revive Media e Preserve Freezing Reagent PathHunter Detection Reagents www discoverx com certified PH_detection_reagen
26. strate a Light f Arrestin EA fi Gal EA Acceptor 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 A ASSAY OVERVIEW Please read the entire protocol completely before running the assay The Assay Procedure sections and Quick Start Guides in this booklet contain detailed information about how to run the assays Refer to the cell line specific datasheet for additional information on the optimized Cell Plating Reagent and reference ligand recommended for the assay Assays should be run using a fresh split of low passage cells that have not been allowed to reach confluency for more than 24 hours Following treatment of the cells with compound GPCR activity is detected by adding a working solution of chemiluminescent PathHunter Detection Reagents using a simple mix and read protocol The following steps are required to monitor GPCR activity using a PathHunter B Arrestin GPCR cell line Figure 2 Plate cells p 9 Dilute and add compounds or antibodies Perform functional assay in agonist p 10 antagonist p 14 or allosteric modulator mode p 18 Plate cells amp Add PathHunter Read add compounds Detection Reagents Luminescence a Figure 2 Simple chemiluminescent assay protocol fo
27. t by combining 1 part Substrate Reagent 2 Substrate with 5 parts Substrate Reagent 1 and 19 parts of PathHunter Cell Assay Buffer entire Plate 384 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 2 Add 12 uL of prepared detection reagent to the appropriate wells DO NOT pipet up and down in the well to mix or vortex shake plates 3 Incubate for 60 minutes at room temperature 23 C Read samples on any standard luminescence plate reader Use GraphPad Prism or other comparable program to plot your agonist dose response See the example shown in Figure 4 REPRESENTATIVE DATA AND DATA ANALYSIS 20000 17500 15000 12500 10000 co 7500 5000 2500 0 10 1310 1210 1110 19 10 9 10 8 10 7 10 6 Somatostatin 28 M Figure 4 PathHunter CHO K1 SSTR2 B Arrestin Cells 93 0181C2 Cells were plated in a 384 well plate at 5 000 cells well and stimulated with the known agonist Somatostatin 28 DiscoveRx 92 1068 for 90 minutes Signal was detected using the PathHunter Detection Kit 93 0001 according to the recommended protocol An assay window of 52 4 fold S B was achieved in this example and the ECsp for agonist was estimated at 1 8 nM 12 QUICK START PROCEDURE AGONIST DOSE RESPONSE Plate 20 uL PathHunter cells well Incubate overnight 37 C Add 5 uL of Agonist Inc
28. th a single ECg concentration of isoproterenol DiscoveRx 92 1119 Signal was detected using the PathHunter Detection Kit 93 0001 according to the recommended protocol 16 QUICK START PROCEDURE ANTAGONIST DOSE RESPONSE Plate 20 uL PathHunter cells well Incubate overnight 37 C Add 2 5 uL of Antagonist Incubate 30 minutes 37 C Add 2 5 uL of Agonist ECgp Incubate 90 minutes 37 C Add 12 uL Detection Reagent Working Solution Incubate 60 Minutes Room Temperature Read Chemiluminescent Signal Please refer to the cell line specific datasheet for any variations in assay conditions 17 ASSAY PROCEDURE ALLOSTERIC MODULATOR DOSE RESPONSE The steps outlined below provide the assay volumes and procedure for performing allosteric modulator assays using PathHunter B Arrestin GPCR Cell Lines and Path Hunter Detection Reagents in a 384 well format Refer to Appendix A for cell numbers and volumes for alternate formats 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 384 well plate ao f T 2 EU i Fa amp oath oo i aa oy 4 Ss gt aF a ps 3 fold serial E amp 3 fold serial E Low dilutions of modulator High wo Low dilutions of mod
29. ts php Microplates www discoverx com certified microplates php PathHunter eXpress B Arrestin www discoverx com gpcrs express_arrestin php GPCR Assays PathHunter eXpress B Arrestin www discoverx com gpcrs express_orphan php Orphan GPCR Assays PathHunter eXpress B Arrestin www discoverx com gpcrs express_ortholog php Ortholog GPCR Assays 28 NOTES 29 NOTES 30 NOTES 31 Contact Information DiscoveRx Corporation World Wide Headquarters 42501 Albrae Street Fremont CA 94538 United States t 510 979 1415 f 510 979 1650 toll free 866 448 4864 KINOMEscan A division of DiscoveRx 11180 Roselle Street Suite D San Diego CA 92121 United States t 800 644 5687 f 858 630 4600 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 www discoverx com DiscoverR DRX UM PH ARRESTIN 0812V1 2012 DiscoveRx Corporation Fremont CA 94538 All rights reserved
30. ubate 90 minutes 37 C Add 12 uL Detection Reagent Working Solution Incubate 60 Minutes Room Temperature Read Chemiluminescent Signal Please refer to the cell line specific datasheet for any variations in assay conditions 13 ASSAY PROCEDURE ANTAGONIST DOSE RESPONSE CURVE The steps outlined below provide the assay volumes and procedures for performing GPCR antagonist assays using the PathHunter B Arrestin GPCR Cell Lines and Path Hunter Detection Reagents in a 384 well format Refer to Appendix A for cell numbers and volumes for alternate formats 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 384 well plate hs Be a amp amp Ey ea Ei o oo a Pa Pa E h oS oe F D D oo os og g 3 fold serial lt gt 3 fold serial moe ie Low dilutions of antagonist High a of Low dilutions of antagonist High 3 ah Is 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 A ci Compound 3 D Ht we Compound 4 Compound 5 L Pea H Compound 6 Compouna Ei esaea ine ae Compound 10 Compound 12 I Compound 9 i PE K Compound 11 SEE BER PEER i UERSRANEANE M E E E EEEE oO aca ee Figure 5 This plate map shows 11 point dose curves with 2 data points at each concentration Plate map
31. ulator High eC 9 10 11 12 13 1415 16 zi 18 19 20 21 22 23 24 7 A E Compound 3 Compound 4 E mi mm EEE eel i m Compound 7 Compound 8 H Ee E Compound 10 Compound 12 A ean ical Be e ppp I Cor d3 ompound 9 TL EEEE K Compound 11 E KH meme aaaea a E o Compound 15 5 HHHH HHHH Compound 16 Figure 7 This plate map shows 11 point dose curves with 2 data points at each concentration Plate map allows 16 modulator compounds to be tested in duplicate per 384 well plate DAY 1 PREPARATION OF ASSAY PLATES Plate PathHunter cells in the appropriate number of wells in a 384 well plate as described in the Preparation of Assay Plates section on p 9 Allow cells to incu bate overnight DAY 2 MODULATOR COMPOUND PREPARATION AND ADDITION 1 Dissolve your allosteric modulator compound in the vehicle of choice DMSO ethanol PBS or other at the desired stock concentration 2 Prepare a series of eleven 3 fold serial dilutions of modulator compound in CP Reagent containing the appropriate solvent DMSO ethanol PBS or other as described below The concentration of each dilution should be prepared at 10X the final screening concentration i e 2 5 uL modulator compound will be used in a final volume of 25 uL For each dilution the final concentration of solvent should remain constant To prepare the 11 point dose curve serial dilutions we recommend starting with a conce
32. yogenic Freezer Vials Fisher Scientific Cat 375418 or similar Multimode or luminescence plate reader Single and multi channel pipettors and pipette tips Tissue culture disposables and plasticware T25 and T75 flasks etc For 96 well analysis we recommend the LumiLITE Microplate Reader DiscoveRx Cat 75 0001 Please refer to the cell line specific datasheet to determine catalog numbers for the media and reagent requirements for the PathHunter B Arrestin cell line you are testing FROZEN CELL HANDLING PROCEDURE To ensure maximum cell viability thaw the vial and initiate the culture as soon as possible upon receipt If continued storage of the frozen vials is necessary store vials in the vapor phase of liquid nitrogen N2 DO NOT store at 80 C for extended periods as this could result in significant loss in cell viability CELL PLATING REAGENT REQUIREMENTS Each PathHunter B Arrestin GPCR cell line has been validated for optimal assay performance using the recommended Cell Plating CP Reagent and control ligand as indicated in the cell line specific datasheet For optimal performance using this PathHunter Certified System always use the CP Reagent recommended for the cell line and DO NOT substitute at any time 6 SOLVENTS AND PREPARATION OF COMPOUND DILUTIONS PathHunter B Arrestin GPCR assays are routinely carried out in the presence of lt 1 solvent i e DMSO ethanol PBS or other As solvents can affe
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