cAMP Hunter™ eXpress GPCR Assay
Contents
1. 10 77 407 10 10 107 10 10 10 10 107 10 10 cAMP M Dopamine M 1 hr read 2 hr read 3 hr read 18 hr read EC 1 40x107 EC 1 96x10 247 d0e 2 53x10 1 82x10 8 S B VA S B 37 6 42 2 43 1 50 4 Glucagon like Peptide Receptor 1 Ga Cells 8000 6000 m Er 4000 aa 2000 0 10 13 10 12 10 11 10 10 10 10 107 GLP1 7 36 M EC 2 2 1x10 50 S B 18 0 DiscoveR US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com 10 CAMP Hunter eXpress GPCR Assay User Manual Appendix Antagonist Protocol for Ga Receptors Protocol Modification 1 Antagonist serial dilution Similar to the agonist step 4a prepare a 10 point series of 3 fold dilutions of antagonist in Cell Assay Buffer The concentration of each dilution should be prepared at 6X of the final screening concentration See plate map below for an antagonist set up example O lt High serial dilutions of antagonist antagonist agonist antagonist agonist cAMP std curve no cells antagonist 1 antagonist 2 antagonist 3 IT O mO OU D gt Antagonist assay plate map Create 10 point curves with cAMP standard curve and 3 different antagonists in duplicate 2 Antagonist Agonist addition Replace step 4b of the agonist detailed protocol with the following directions a Add 7 5 ul of each 6X antagonist serial dilution to the designated antagonist r2. 6 CAMP Hunter eXpress GPCR Assay User Manual No agonist control A cAMP std curve no cells B C agonist 1 D E agonist 2 F G agonist 3 H Agonist assay plate map Create 11 point curves for cAMP standard curve and 3 different agonists in duplicate Step 2 Assay Plate Preparation i ae Removing the media completely a Completely remove the cell media from assay plate wells by aspiration is crucial for reducing variability of replicates b Immediately add 30 uL of Cell Assay Buffer to all empty wells in the assay plate Step 3 cAMP Standard Preparation When optimizing the assay conditions always include a cAMP standard curve The standard curve not only verifies that the kit components are working properly but also serves as a detection limit guide If the amount of cAMP being detected exceeds the detection limit of the cAMP detection kit the EC will start to shift depending on the coupling status of Ga or Ga the shift will be towards the right or left To avoid this situation the cell number per well should be optimized cAMP standard should be prepared fresh before agonist compound addition a Prepare cAMP standard serial dilutions in a separate plate by diluting the cAMP standard 2 5 x 10M in a 1 9 ratio so 1 part cAMP standard plus 8 parts Cell Assay Buffer This dilution ratio corresponds to the highest standard concentration well No 1 at 2 31 x 10 M in an assay volume of 180 uL Make 10 a
3. performance of the cells and detection reagents Protocol Modification 1 Replace step 4a of the agonist detailed protocol with the following steps to D For antagonist Go coupled l nae receptors forskolin is added to prepare a Ga coupled receptor ligand plus forskolin 11 point serial dilution the agonist challenge solution a Prepare a 3X for agonist mode or 6X for antagonist mode final screening concentration of forskolin Refer to the target specific datasheet for recommended concentrations of forskolin A general recommendation A eh eee reeze thaw cycles Store as for the final forskolin concentration is 10 15 uM for CHO K1 cells and multiple aliquots at 20 C for 15 20 uM for HEK 293 For example if the forskolin final in well screening up to 2 months concentration is recommended to start at 15 uM prepare a 45 uM forskolin solution which is 3X b Prepare the highest concentration 3X for agonist mode or 6X for antagonist mode of Ga agonist in the forskolin solution prepared above c Add 120 uL of the highest concentration of agonist plus forskolin to well No 1 d Remove 40 uL from well No 1 and add it to well No 2 Mix gently e With clean tip remove 40 uL from well No 2 and add it to well No 3 Mix gently f Repeat process until well No 11 Do not add agonist to well No 12 since this is the negative control well g Set up additional serial dilutions for additional agonists in a similar man
4. uL 80 uL 80 uL 80 uL 80 uL 80 uL 120 uL 80 uL Sequential Dilution Ratio 1 9 1 3 1 3 1 3 1 3 1 3 1 3 1 3 1 3 1 3 1 3 Final Assay Concentration M 2 31x10 7 70x107 2 57 x107 8 56x108 2 85x108 9 51x10 3 17x10 1 06x10 3 52x10 1 17 x10 3 91 x10 cAMP Standard serial dilutions Make 11 three fold serial dilutions of cAMP standard solution in a separate plate Step 4 Agonist Preparation of 3X 3 fold dilutions of agonist in Cell Assay Buffer or appropriate agonist ligand variations e g antagonists buffer as specified on the datasheet The final concentration of each dilution a le ap PenonsSectan should be prepared at 3X of the final screening concentration a Prepare agonist serial dilutions in a separate dilution plate in a 11 point series D For Ga specific targets or other 1 For each agonist label the wells of a separate dilution plate or polypropylene tubes No 1 to No 12 2 Similar to the cAMP standard serial dilutions add 80 uL of Cell Assay Buffer or agonist specific buffer to dilution wells No 2 to No 12 This is enough volume for over 4 rows and the dilution volume may be adjusted according to the number of wells desired 3 Prepare the highest concentration of Agonist in Cell Assay Buffer or agonist specific buffer We recommend preparing a final screening concentration that is 250X the expected EC of the agonist Therefore prepare a working concentration that is 750X the expected EC per wel
5. MSO and other solvents are not too high and not more than 1 final concentration e When testing ligands at high concentrations make sure the vehicle is compatible e Make sure ligand is incubated for the designated time and at designated temperature e Make sure plates are protected from light during incubation What if the EC does not match reported values e Include a cAMP standard curve in the assay to ensure that the ligand dose curve is in the dynamic range of the cAMP detection kit Moving out of the dynamic range may shift the EC of ligands e Make sure ligands are incubated at the proper temperature e The source of a ligand particularly for biologics can have a large effect on the observed pharmacology e Changing tips during serial dilutions can help to avoid carryover e Receptor expression level may cause receptor reserve issues in ligand testing Select a cell line that has medium to low expression of receptors What if the variability between replicates is high e Make sure dispensing equipment is properly calibrated and proper pipetting technique is used What if my compound is in media containing high concentrations of serum can I use it as is or will the serum interfere with the assay e Our assays are highly tolerant to high serum content as high as 80 serum We recommend that you aspirate the high serum media prior to adding detection reagents For additional information or Technical Support contact info dis
6. Reserved DiscoveRx logo and DiscoveRx are registered trademarks of DiscoveRx Corporation 20370 041615 DiscoveR US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com CAMP Hunter eXpress GPCR Assay User Manual Overview The cAMP Hunter eXpress GPCR Assay can be used for small or large molecules The kits provide a robust highly sensitive and easy to use assay for monitoring G protein coupled receptor GPCR activation based on 3 5 cyclic adenosine monophosphate cAMP production in cells Technology Principle GPCR activation following ligand binding initiates a GPCR cAMP Pathway series of second messenger cascades that result in a cellular response The signaling involves a membrane bound enzyme called adenylate cyclase Ga and Ga coupled receptors modulate cAMP by either inhibiting or stimulating adenylate cyclase respectively With the CAMP Hunter eXpress GPCR Assay cells over expressing GPCRs utilize the natural coupling status of the GPCR to monitor activation of Ga and Ga coupled receptors Following ligand stimulation the functional status of the GPCR is monitored by measuring the cellular cAMP levels using a homogeneous no wash gain of signal competitive immunoassay based on Enzyme Fragment Complementation EFC technology The cAMP Hunter eXpress GPCR Assay EFC technology uses a B galactosidase B gal enzyme split into two fragments the E
7. a Following agonist incubation add 15 uL of cAMP Antibody Reagent to all wells Make stock within 8 hours of use b Prepare a stock of cAMP Working Detection Solution in a separate 15 ml polypropylene tube by mixing cAMP Working Detection Solution 19 parts of cAMP Lysis Buffer 5 parts of Substrate Components Volume ratio Volume per plate Reagent 1 1 part Substrate Reagent 2 and 25 parts CAMP Lysis Buffer 19 3 8 ml of cAMP Solution D Substrate Reagent 1 5 1 0 mL E E E PE T T E E E E Substrate Reagent 2 1 0 2 mL A orang erection souton toa PS 25 5 0 mL wells of the assay plate Do not pipette up and down i Total Volume 10 mL in the vial to mix or vortex plates d Incubate assay plate for 1 hour at room temperature Q cAMP Working Detection Solution is light sensitive thus in the dark for the immunocompetition reaction to incubation in the dark is necessary occur Step 6 Enzyme Acceptor Addition a Add 60 uL of cAMP Solution A to all wells of the assay plate Do not pipette up and down in the vial to mix or vortex plates b Incubate assay plate for 3 hours at room temperature in Q cAMP Working Detection Solution is light sensitive thus the dark incubation in the dark is necessary Step 7 Assay Plate Reading a Read samples on a standard luminescence plate reader at 0 1 to 1 second well for photomultiplier tube readers or 5 10 seconds for imager The plate may be incubated overnight and the signal may be measur
8. ancien ns nae sce ce eave nce Rin ee tented cele meeweatn che nentrn enero es 8 Step 5 Antibody and Working Solution Addition cccccccceececesceceeeeceececeeeceaeeceeeeseeesseeesaueesaueesaueessueessueessueessaees 9 Step C Enzyme Acceptor AQQIUON sisses orisii ia E A o E E ei 9 Be FS ay Plate Reading errors Eene n eu ewete dae estrues ens tomatuetentdonebiesteccaeetenendssicesyancesuerduanccaueeedantetanruddeccbumedenersoan 9 WY POUC AN FRCS CS ceca aE E 10 APPENA o E 11 Antagonist Protocol Tor Ga Coupled Receptors serrer Aa aeaa aa 11 Protocolior GG COUDIEG RECEPTIONS iseproroisr piesists ansia pE ri E E REENE r POENE EN ENEA EENE ERE E 12 PANS ONG NAMO inn EEA E A TEE NEE E ESSES 13 Hion IProugnput SeraSNilig srersrreroersi rene mere tuerienr ryt etre ereoner Mecar aatrr etree er ele treet ner eer eet eee ere eee 13 Crude Biologic Samples sssr eee rte Sart recige tee ee cated Sane st naw ents ie enced E EE eT 13 Adjusted Volumes for 384 well Format cectssic accent coinga vasmeanioe sou tanctinn seeiiaciacatiedneesiebusesessiquslndaeseuauueescaacuubnenencelGencimbelaneesens 13 PAOS ace cra cheater ease cee teem ee eens eee a 14 Limited Use License AGlOG SU siiiceiatasccsanasnnpseastmecteasntnaitusisenmanmusnsassneaspsaneus AENEA AANA 15 Please read entire booklet before proceeding with the assay For additional information or Technical Support contact info discoverx com or visit www discoverx com 2015 DiscoveRx Corporation All Rights
9. cAMP Hunter eXpress GPCR Assay For chemiluminescent detection of cAMP for GPCR activity Simple Solutions for Complex Biology DiscoverR Table of Contents OVER VOW atcusnneccatesasisanenenecacesnpesiuseapwacavesapsnacwatnaycanedesnaaunsavadsanccaiesaunasusoainesnavaveaevaaareusvanacadsaibecisuciwauedadsceunbasianvaneesdiescieu 1 Technology FENG UON ranieva cence imandecuudnneiuciionnweanthiu na cod caduuanedtiaidedneydusines AEAEE EAE EA EEEE AAN 1 UNE SC USG oina S E alscow pied am ve aan ane eme a 2 Materials Provided and Storage Conditions scccsccenscenseensscenseensecnsecenseonsconseensseonseoessonsseoasennssceasensssnnsennsssessenaes 3 Additional Materials sisanra iann a aa aa A a ia A Aaa a a A aAA a A aE 3 PROTOCOl SCHEIN ANIC irirna 5 NUTS UG POCO S seapea 5 Detailed Protocol Agonist 96 well Ga sssssssussnunnennnnnunnnnunnnnunnnnnnnnnnnnunnnnunnnnnnnnnnnnnnnnnnnn nnnm mnnn nananana nnmnnn nananana nna 6 Step 1 Cell Preparation waccdurccunnessdeareeuskscnuvensuectinneniue cetoepihdssdeanabuesmectentiecumonddedranvocaiteruteonasiecumauaneucerahiestapenmulaumocane 6 Step 2 ASSay Plate Preparato iene hin etacctiotnnoaetionteanienetmanenisanieictanstion E ERa as ers ene aE SEn raii dnia iesenii eneas T otep 9 CAMP Standard IPPC Dal AON scrissero ssuecuasteieductestenencdasttennaadectescesmenssabictatentdusnedenctantecunnioeentsses T Step 4 Agonist Preparation a cae ci crs tats ere se ce rec ceed gee cts cecre i
10. coverx com or visit www discoverx com DiscoveR US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com 14 CAMP Hunter eXpress GPCR Assay User Manual Limited Use License Agreement DiscoveRx name logo and cAMP Hunter are trademarks of DiscoveRx Corporation DiscoveRx grants to the purchaser of this product an end user license under United States and foreign patents owned or licensed exclusively to DiscoveRx including US patent numbers 4708929 4956274 5244785 5444161 5604091 5643734 and equivalent foreign patents and non exclusively licensed including US patent numbers 4931569 4978614 5145772 5326882 5538847 and 5851771 and equivalent foreign patents to use this product solely in the fields of drug discovery and regulatory qualification of therapeutic drugs and not for any other uses including but not limited to other research uses or use in human clinical animal or food diagnostics If the purchaser 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 t 510 979 1415 x 104 e info discoverx com cAMP Hunter eXpress GPCR Assay User Manual 70 234 Rev_8 D m ISCOVER US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com 15
11. dditional 3 fold serial dilutions using the Cell Assay Buffer with the last well as the negative control as shown below 1 Using a separate dilution plate or polypropylene tubes label the wells No 1 to No 12 2 Add 160 uL of Cell Assay Buffer and 20 uL of cAMP standard to well No 1 this will be the highest concentration of cAMP standard 3 Add 80 uL of Cell Assay Buffer to dilution wells No 2 to No 12 This is enough volume for more than 4 rows 4 Remove 40 uL from well No 1 and add it to well No 2 Mix gently 5 With clean tip remove 40 uL from well No 2 and add it to well No 3 Mix gently DiscoveR US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com T CAMP Hunter eXpress GPCR Assay User Manual 6 Repeat this process until well No 11 Do not add cAMP standard to well No 12 since this is the negative control well containing only Cell Assay Buffer b Add 15 uL of cAMP standard dilutions in duplicate to the first two rows rows A and B of the assay plate as shown in the previously described assay plate map cAMP 20 pL Standard 40L 40uL 40 uL 40 uL 40 uL 40 uL 40 uL 40 uL 40 uL 40 uL Negative Control gt lt l DX See J Cell ZAA x L lt OOOO C Assay L L L L i 7 no CELY CILY CIT CITY CITY CI UI T TP 1 2 3 4 5 6 T 8 9 10 11 12 Final Volume 140 uL 80 uL 80 uL 80 uL 80
12. e of IBMX If IBMX is to be used cell number per well and IBMX concentration needs to be optimized so that the amount of cAMP generated stays within the optimal detection of the assay kit How do I use suspension cells e Harvest and resuspend suspension cells in Cell Assay Buffer media either 1X HBSS 10MM HEPES or PBS at the optimal cell density Typical suspension cell density is approximately 20 000 cells per well in a standard 96 well plate with a cell viability gt 90 What if there is no or low signal e f plated on clear bottom assay plates visually inspect the cells before and after running the assay to ensure that they are healthy Make sure proper cell plating procedure is followed to ensure assay performance e Make sure detection reagents are stored and prepared properly e Make sure forskolin is included for Ga coupled receptors Please refer to the Appendix for Ga coupled receptor assay protocol e Make sure the proper assay mode is used agonist mode or antagonist mode e Make sure the plate reader is setup properly to read luminescence e High levels of protein in the wells may interfere with enzyme fragment complementation If high levels are present a media exchange step could be performed just prior to the detection reagent addition What if the response is lower than expected lower than expected S B e Make sure that the ligand is prepared properly take extra care to observe ligand solubility e Make sure D
13. ed the next day In general the signal continues to increase and reaches a maximum approximately 3 6 hours after the last incubation step The actual signal characteristics over time are affected by lab conditions such as temperature and the user should establish an optimal read time Luminescence readout usually collects signal from all wavelengths Some instrument manufacturer may include a cutoff filter at high wavelengths but usually no wavelength setting is needed for luminescence readout b Data analysis can be performed using your choice of statistical analysis software e g GraphPad Prism Molecular Devices Softmax Pro Biotek Instruments Gen5 Microsoft Excel etc DiscoverR US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com 9 CAMP Hunter eXpress GPCR Assay User Manual Typical Results Typical results shown below of the cAMP standard curve analysis top left and cAMP Hunter eXpress GPCR Assay using the cAMP Hunter dopamine receptor D2 short isoform Ga cells top right and glucagon like peptide receptor 1 Ga cells bottom Note for the cAMP standard curve graph the signal continues to increase over time and plates can be read the following day cAMP Standard Curve 96 well Dopamine Receptor D2 20000 short isoform Ga Cells e hr read 0000 2hr read 8000 i Shr read 18 hr read gt 6000 10000 l 4000 one 2000 0 0
14. ends storing the vials in the vapor phase above the liquid N Upon thawing if liquid N is present in the cryovial it rapidly converts back to its gas phase which can result in the explosion of the vial upon its removal from the freezer Required Materials Disposable Reagent Reservoir Green V bottom Ligand Dilution Plates 10 plates pack 15 mL polypropylene tubes and 1 5 mL microtubes Tissue culture disposables pipettes 1 mL 25 mL Humidified tissue culture incubator 37 C 5 CO Single and multichannel micro pipettors and pipette tips 10 uL 100 uL Multimode or luminescence plate reader US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 Ordering Information Thermo Scientific Cat No 8094 or similar DiscoveRx Cat No 92 0011 DiscoveR e euroinfo discoverx com www discoverx com 3 CAMP Hunter eXpress GPCR Assay User Manual Recommended Materials Reagents and Equipment Ordering Information Control ligands www discoverx com controlligands Test Compounds IBMX for Ga assays DiscoveRx Cat No 92 0007 Instrument Compatibility Chart Compatible with any Luminometer Select examples indicated below Berthold Technologies Mithras LB940 CentroLlApc Molecular Devices FLIPR SpectraMax M3 M4 M5 M5e FlexStation 3 SpectraMax L Biotek Synergy 2 Perkin Elmer TopCount Victor 2 or V Fusion LumiCount Envision Micro beta Trilux Viewlux Northstar EnSpire BMG Phe
15. f the standard protocol Crude Biologic Samples The cAMP Hunter eXpress GPCR kits can be run in the presence of high levels of serum or plasma without significantly impacting assay performance Therefore standard curves and samples can be prepared in neat serum or plasma and added directly to cells without further dilution For the best results the optimized minimum required dilution MRD of crude samples should be empirically determined Adjusted Volumes for 384 well Format Use the following table to adjust the component volumes per well for 384 well plates 384 well Format Assay Reagents 96 plate per well 384 plate per well No of Cells 30 000 10 000 AssayComplete Cell Plating Reagent 100 uL 20 uL Cell Assay Buffer 30 uL 10 uL Ligand e g Agonist 15 uL 5 uL cAMP Antibody Reagent Io uk 5 uL cAMP Working Detection Solution 60 uL 20 uL cAMP Solution A 60 uL 20 uL Final Assay Volume 180 uL 60 uL The number of cells is dependent on the cell line The AssayComplete Cell Plating Reagent volume is used to plate cells and this volume is removed during first step DiscoverR US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com 13 CAMP Hunter eXpress GPCR Assay User Manual FAQs Is IBMX necessary in the DiscoveRx cAMP kits e IBMX is not necessary and we do not use it for our internal testing however the kit is compatible with the us
16. hase which can result in the explosion of the vial upon its removal from the freezer b Decontaminate the vial by spraying and wiping with 70 ethanol All of the procedures from this point on should be carried out under aseptic conditions in a culture hood c Pre warm Assay Complete Cell Plating Reagent in a 37 C water bath for 15 mins d Add 0 5 mL of pre warmed AssayComplete Cell Plating Reagent to the Donoi thawviae nar C waierbath cell vial to thaw the cells Pipette up and down gently several times to or centrifuge ensure that cells are evenly distributed e Immediately transfer the cells to 11 5 mL of pre warmed AssayComplete Cell Plating Reagent Mix and pour into a disposable reagent reservoir f Prepare the 96 well tissue culture treated assay plate using the plate map figure on the next page Leave the first 2 rows empty with no cells to allow for an 11 point cAMP standard curve in duplicate For the remaining wells plate 100 ul of cells into each well For rows C through H plate 3 different 11 point agonist curves in duplicate Optionally samples can be run on different plates in triplicates or other variations g Place the seeded plate in a 37 C 5 CO humidified incubator for 18 24 hours prior to testing refer to the target specific datasheet for the recommended incubation time DiscoveR US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com
17. he dark Add 60 uL CAMP Solution A Incubate for 3 hrs at room temp in the dark Read chemiluminescent signal DiscoveR US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com 5 CAMP Hunter eXpress GPCR Assay User Manual Detailed Protocol Agonist 96 well Ga The following detailed protocol is specific to detecting cAMP in cells D Refer to the appendix for variations stimulated by an agonist for Ga coupled receptors in a 96 well format plate e g agonist Ga coupled antagonist HTS PAMs NAMs crude biologics and 384 well Step 1 Cell Preparation Do not substitute cell plating from an CAMP Hunter eXpress cells from cryogenic vials Each vial contains alternate kit at any time sufficient cell numbers to generate 1 96 well microplate prepared at the seeding density described The following steps outline the procedure for thawing and plating frozen D Do not expose vials to room liquid N vapor storage and place them immediately on dry ice prior to temperature a Remove the cAMP Hunter eXpress Assay cells cryovials from 80 C or ae thawing Safety Warning A face shield gloves and lab coat should be worn at all times when handling frozen vials The manufacturer of the cryovial recommends storing the vials in the vapor phase above the liquid N Upon thawing if liquid N is present in the cryovial it rapidly converts back to its gas p
18. l E g for an expected EC of 1 nM prepare the highest working concentration at 750 nM This is 3X the screening or final top concentration of 250 nM and the expected EC concentration will lie near the center of the dose response curve 4 Add 120 uL of the highest concentration of Agonist Cell Assay Buffer to well No 1 5 Remove 40 uL from well No 1 and add it to well No 2 Mix gently 6 With clean tip remove 40 uL from well No 2 and add it to well No 3 Mix gently 7 Repeat process until well No 11 Do not add agonist to well No 12 since this is the negative control well 8 Setup additional serial dilutions for additional agonists in a similar manner DiscoveR US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com 8 CAMP Hunter eXpress GPCR Assay User Manual b Add 15 uL of each 3X agonist serial dilution in duplicate to the designated agonist rows e g agonist 1 in rows C and D agonist 2 in rows E and F and agonist 3 in rows G and H of the assay plate as indicated on the previously described assay plate map c Incubate assay plate at the indicated time and temperature for the specific cell line please refer to the specific cell line datasheet for conditions For most cell lines incubate for 30 minutes at 37 C For the best results the optional incubation time should be empirically determined Step 5 Antibody and Working Solution Addition
19. me in each bottle mL Storage cAMP Lysis Buffer 3 8 7 6 38 Upon arrival store reagents at lt 20 C The detection kit is CAMP Solution D 5 10 50 stable until the expiry date indicated on the kit box outer label ANP Solution A Thaw reagents at room temperature before use After thawing CEREN 8 16 80 store reagents for up to 1 month at 2 8 C For longer term cAMP Antibody Reagent 2 9 5 29 storage aliquots of all the components may be re frozen in cAMP Standard 250 uM 1 1 1 opaque containers at lt 20 C The reagents can be thawed and Substrate Reagent 1 1 D 10 frozen for a total of 3 times without loss in performance Substrate Reagent 2 0 2 0 4 2 Forskolin dried powder 1x 0 25 2 x 0 25 10x 0 25 If Forskolin is not used immediately prepare multiple aliquots mg mg mg and store at 20 C Avoid multiple freeze thaw cycles When stored properly the solution is stable for up to 2 months Cell Assay Buffer Ze 25 250 20 C Assay Complete Cell 1x20mL 2x20mL 2x100mL 20 C Plating Reagent Plate Format 1 plate 2 plates 10 plates Room Temperature 96 well No of data points 100 200 1 000 Included 96 well white walled clear bottom tissue culture treated plate 384 well No of data points 400 800 4 000 Not Included Bulk cells are available upon request Additional Materials Safety Warning A face shield gloves and lab coat should be worn at all times when handling frozen vials The manufacturer of the cryovial recomm
20. ner 2 Replace step 4b of the agonist detailed protocol Add 15 uL for agonist mode or 7 5 uL for antagonist mode of the Ga agonist plus forskolin serial dilutions in duplicate to the designated agonist rows e g agonist 1 in rows C and D agonist 2 in rows E and F and agonist 3 in rows G and H of the assay plate as indicated on the previously described assay plate map 3 Continue with step 4c incubation of the agonist detailed protocol DiscoverR US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com 12 CAMP Hunter eXpress GPCR Assay User Manual PAMs and NAMs For positive allosteric modulators PAMs refer to the antagonist protocol above but change the agonist challenge concentration to EC instead of EC For negative allosteric modulators NAMs refer to the antagonist protocol above with no changes High Throughput Screening For high throughput screening applications the cAMP Antibody Reagent can be added before ligand introduction to save an addition step Briefly pre dilute the cAMP Antibody Reagent from the kit in a 1 2 ratio with Cell Assay Buffer So 1 part cAMP Antibody Reagent to 2 parts Cell Assay Buffer Replace step 2a of the agonist detailed protocol by adding 45 ul of diluted antibody mixture to each well Replace step 4b by adding 15 ul of 4X agonist to each well Incubate the assay plate as indicated in step 4c and skip to step 5b o
21. nzyme Donor ED and Enzyme Acceptor EA Independently these fragments have no gal activity however in solution they rapidly complement to form an active B gal enzyme In this assay cAMP from cells and ED labeled cAMP ED cAMP compete for anti cAMP antibody Ab binding sites Antibody bound ED cAMP will not be able to complement with EA but unbound ED cAMP is free to complement EA to form active enzyme which subsequently produces a luminescent signal The amount of signal produced is directly proportional to the amount of cAMP in the cells Low Levels of Cellular cAMP 4 y a gt ve r B gt YN 83 Cellular cAMP Anti cAMP Ab ED cAMP EA High Levels of Cellular cAMP f y x Substrate 7x Hydrolysis 30 Yy gt J 93 l Active EFC Light Cellular cAMP Anti cAMP Ab ED cAMP Enzyme Discover US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com 1 CAMP Hunter eXpress GPCR Assay User Manual Intended Use The cAMP Hunter eXpress GPCR Assay kits provide a robust highly sensitive and easy to use cell based functional assay to study GPCR activity through cAMP production The pre validated frozen eXpress cells have been manufactured for short term use and are provided in a convenient ready to screen format that saves time and adds convenience The kits contain all the reagents needed for the detection of cAMP from eXpress cells expressing Ga and Ga cou
22. ows of the assay plate b Incubate assay plate for 15 minutes at 37 C For best results the optimal incubation time should be empirically determined c For the agonist challenge add 7 5 uL of agonist at 6X the EC p For Ga targets only add the forskolin during the agonist We recommend running the agonist dose response curve to determine the challenge step EC The specific concentration of agonist to be used can vary from EC to EC based on the target and assay conditions For Ga targets instead of the agonist alone add 7 5 uL of 6X forskolin plus agonist solution at this time d Continue with step 4c incubation of the agonist detailed protocol DiscoveR US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com 11 CAMP Hunter eXpress GPCR Assay User Manual Protocol for Ga Coupled Receptors In order to measure Ga coupled receptors targets the agonist sample is added in the presence of forskolin Forskolin activates adenylate cyclase and increases intracellular levels of cAMP For a Ga receptor agonist binding or activation inhibits the intracellular cAMP accumulation induced by forskolin As a result the dose response curve generated in the presence of agonist plus forskolin will have a negative slope Since forskolin treatment results in increased intracellular CAMP levels in all CAMP Hunter cells tt can serve as a positive control for the
23. pled receptors induced with a small or large molecule ligand This flexible assay system has been designed to work in agonist or antagonist mode for 96 and 384 well plate formats After plating and stimulation of cells the user simply adds the assay reagents to the cells following the homogeneous easy to use protocol provided cAMP Hunter eXpress GPCR Assay Schematic Protocol Plate Cells Working esc Antibody Assay ED cAMP Solution Solution EA Read signal Buffer Ligand 0 9 o E ge Incubate Incubate y y Discover US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com 2 CAMP Hunter eXpress GPCR Assay User Manual Materials Provided and Storage Conditions List of Components Cell Components Number of cells Storage CAMP Hunter eXpress Cells 1 vial 2 vials 10 vials Cells are shipped on dry ice and should arrive in a frozen state Number of Cells 375x10 3 75x10 3 75x10 TO ensure maximum cell viability thaw the vials as soon as cells each cells each cells each possible upon receipt If continued storage of the frozen vials vialin 200 vialin200 vialin200 S necessary store vials at 80 C immediately upon arrival DO uL uL uL NOT store at 80 C for gt 2 weeks For longer term storage gt 2 weeks store vials in the vapor phase of liquid nitrogen N DO NOT store vials in liquid phase of N see Safety Warning below Kit Components Volu
24. raStar Cytostar LumiStar Promega GloMax systems Caliper LabChip 3000 amp EZ Reader Tecan Ultra Evolution GE LEAD seeker Farcyte Thermo Scientific Luminoskan Ascent Hamamatzu FDS6000 FDSS RayCatcher Turner BioSystems Modulus Microplate DiscoveR US tel 1 866 448 4864 e info discoverx com EU tel 44 121 260 6142 e euroinfo discoverx com www discoverx com 4 CAMP Hunter eXpress GPCR Assay User Manual Protocol Schematic Tip Use this sheet to note your assay specific Assay Name Date conditions Post on your bench to use as a quick reference guide Product Details Quick Start Procedure In a white walled 96 well tissue culture treated plate perform the following Assay Preperation cAMP Hunter eXpress GPCR Detection Seed cells in 100 uL Agonist Cell Plating Reagent Antagonist Incubate cells overnight at 37 C 5 CO Remove Cell Plating Reagent Replace with 30 uL Cell Assay Buffer Treat cells with 7 5 uL 6X Antagonist prepared in Cell Assay Buffer Incubate for 15 mins at 37 C Treat cells with 7 5 uL 6X Agonist prepared in Cell Assay Buffer Include 6X forskolin for Ga targets Treat cells with 15 uL 3X Agonist prepared in Cell Assay Buffer Include 3X forskolin for Ga targets Incubate for mins at C Specific time amp temp in datasheet Add 15 uL Antibody Solution Add 60 uL CAMP Working Detection Solution Incubate for 1 hr at room temp in t
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