LANCE® Ultra cAMP Kit
Contents
1. Important Product News Date February 21 2011 Product Change Notification Dear Valued Customer Upon reception of the LANCE Ultra cAMP kit please store the Eu cAMP tracer aliquoted and frozen at 20 C We recommend that you limit the number of freeze thaw cycles of the tracer All other kit components should be stored at 4 C For additional information please consult the user manual Pp alee ir LANCE Ultra cAMP Kit For Research Use Only o 1 Intended use The LANCE Ultra cAMP kit is intended for the quantitative determination of 3 5 cyclic adenosine monophosphate cAMP in cell lysate and cellular membrane samples 2 Provided reagents Component TRFO262 TRFO263 TRFO264 1 000 points 10 000 points 50 000 points cAMP standard 50 uM 1 vial 1 mL 1 vial 1 mL 1 vial 1 mL Eu cAMP tracer 1 vial 110 uL 1 vial 1 mL 5 vials 1 mL each ULight anti cAMP 1 vial 37 uL 1 vial 340 uL 1 vial 1 68 mL cAMP Detection Buffer 1 bottle 25 mL 1 bottle 250 mL 4 bottles 250 mL each BSA Stabilizer 7 5 solution 1 vial 1 mL 1 bottle 10 mL 1 bottle 50 mL When using the recommended protocols 20 uL assay in 384 well microplates Centrifuge tubes for a few seconds before use to improve recovery of content Store the Eu cAMP tracer aliquoted and frozen at 20 C Avoid repeated freeze thaw cycles 3 Storage conditions Upon receiving the kit store the Eu cAMP tracer aliquote2. E E 7 Lu 10 000 w 5 0004 fc 250 i i cc cc E 0 r T T E 042 tH T T T T T on 11 10 9 8 7 6 5 11 10 9 8 7 6 5 Log cAMP M Log cAMP M EnVision EnVision Laser Lamp Max counts 24178 16352 30440 1366 VICTOR ViewLux Min counts 356 417 638 68 S B 67 9 39 2 47 7 20 1 ICs nM 1 42 1 42 1 45 1 32 Figure 3 Representative LANCE Ultra cAMP standard curves obtained on different instruments using the recommended settings A white OptiPlate 384 microplate with a single cAMP standard curve assay was incubated for 1 hour at room temperature and then read with the A EnVision Multilabel reader laser and lamp settings VICTOR reader and B ViewLux NOTE Depending on the instrument counts and S B ratio may vary but this will not affect significantly assay robustness or sensitivity ICso 11 Assay volumes recommended for different plate formats AreaPlate 96 OptiPlate 384 OptiPlate 1536 Cat 6005560 Cat 6007290 Cat 6004290 20 uL 8 uL See instrument settings in Section 9 For technical application assistance please contact PerkinElmer technical support PerkinElmer Life and Analytical Sciences Direct Dial U S 800 762 4000 Toll Free Europe 00800 33290000 For Finland please dial 999 800 33 29 0000 E mail global techsupportO perkinelmer com Please visit www perkinelmer com for specifi
3. IBMX to the Stimulation Buffer is optional For cell and membrane based assays IBMX could be replaced by another phosphodiesterase inhibitor e g 100 uM RO 201724 Addition of BSA might not be essential for your cellular assay However if BSA is used we strongly recommend the BSA Stabilizer 7 5 solution included in the kit as it is a highly purified preparation of BSA free of europium and heavy metal ion contaminants 7 2 cAMP standard serial dilutions in Stimulation Buffer Prepare the 4X cAMP standard serial dilutions in Stimulation Buffer from the 50 uM cAMP standard supplied with the kit as indicated in the table below Dilution Final M 4X M Volume of dilution Stimulation Buffer a 1x10 4X 10 8 uL of 50 UM cAMP 92 uL 3X10 1 2 X 10 30 ul of 1 70 uL 3 1x0 4x10 30 uL of 2 60 uL 4 3x10 1 2 X 10 30 uL of 3 70 uL 5 1x10 4x 10 30 uL of 4 60 uL e Sao taxi 30utofs w Pe oo aaxi owo Fo o io axi0 owo oom ao NET GTI 22x10 SoWtore zo an a axo somero eo Pie a 7 3 Eu cAMP tracer solution in cAMP Detection Buffer Prepare a 4X Eu cAMP tracer working solution by making a 1 50 dilution of the Eu cAMP tracer stock solution in cAMP Detection Buffer Example Add 5 uL of the Eu cAMP tracer stock solution to 245 uL of cAMP Detection Buffer and mix gently 7 4 ULight anti cAMP solution in cAMP Detection Buffer Prepare a 4X ULight anti cA
4. MP working solution by making a 1 150 dilution of the ULight anti cAMP stock solution in cAMP Detection Buffer Example Add 5 uL of the ULight anti cAMP stock solution to 745 uL of cAMP Detection Buffer and mix gently NOTES e Working solutions can be stored up to 24 hours at 4 C e For optimal assay performance do not modify the recommended dilutions for both the Eu cAMP tracer and ULight anti cAMP 8 Assay protocols for a 384 well plate total assay volume of 20 uL In the protocols described in the table below both the cells and tested compounds must be prepared in Stimulation Buffer including 0 5 mM IBMX cAMP Detection Buffer must be used only for the preparation of Eu cAMP tracer and ULight anti cAMP working solutions la Gs Agonist Gs Antagonist g Forskolin Gi Agonist Gi Antagonist curve titration 5 pL cAMP 5 pl cell 5 pl cell 5 uL cell 5 pl cell 5 pl cell standard suspension suspension suspension suspension suspension an 2 5 uL Stimulation 5 pl Agonist 2 5 uL Agonist 5 uL Forskolin 2 5 uL Forskolin H Forskolin Agonist Buffer 2 5 uL 3 2 5 uL Agonist 2 5 uL Antagonist Antagonist Hr 58 H 8 Incubate 30 min at room temperature optional step for cAMP standard curve 5 pL 4X Eu cAMP tracer working solution 5 pL 4X ULight anti cAMP working solution Incubate 1 h at room temperature Read on a TR FRET microplate reader Remove microplate seal prior to reading Cover microplate w
5. c country contact details PerkinElmer Inc 940 Winter Street I Waltham MA 02451 USA _ Phone 800 762 4000 or Perkin 1 203 925 4602 www perkinelmer com e For the Better 2010 PerkinElmer Inc All rights reserved The PerkinElmer logo and design are registered trademarks of PerkinElmer Inc EnVision ViewLux and LANCE are registered trademarks of PerkinElmer Topseal A ULight and Victor are trademarks of PerkinElmer Other trademarks are the property of their respective owners The ULight dye is claimed in PCT Application No PCT US2010 021282 and equivalents PerkinElmer reserves the right to change this document at any time without notice and disclaims liability for editorial pictorial or typographical errors TRFO262 TRFO263 TRFO264 v6 Printed in USA 8
6. d and frozen at 20 C and all other reagents at 2 8 C protected from light The expiration date of the kit is indicated on the box label 4 Assay principle The LANCE Ultra cAMP assay is a homogeneous time resolved fluorescence resonance energy transfer TR FRET immunoassay designed to measure cAMP produced upon modulation of adenylyl cyclase activity by G protein coupled receptors GPCRs The assay is based on the competition between the europium Eu chelate labeled cAMP tracer and sample cAMP for binding sites on cAMP specific monoclonal antibodies labeled with the ULight dye Figure 1 When antibodies are bound to the Eu labeled cAMP tracer light pulse at 320 or 340 nm excites the Eu chelate molecule of the tracer The energy emitted by the excited Eu chelate is transferred by FRET to ULight molecules on the antibodies which in turn emit light at 665 nm Residual energy from the Eu chelate will produce light at 615 nm In the absence of free cAMP maximal TR FRET signal is achieved Figure 1 left panel Free cAMP produced by stimulated cells competes with the Eu cAMP tracer for the binding to the ULight mAb causing a decrease in TR FRET signal Figure 1 right panel In the absence of free cAMP In the presence of free cAMP Excitation TR FRET 3200r340nm Emission Excitation a a ya No FRET 320 or 340 nm Fluorescent E Emission de wp 615nm Fluorescent Free cAMP Emission 615nm Figure 1 LANCE Ultra cAMP assay princi
7. ith a TopSeal A film PerkinElmer Inc Cat 6005250 or another plate during incubations NOTES e Additional readings can be performed for at least 24 hours after addition of LANCE Ultra reagents without significant change in assay sensitivity e If preferred in order to eliminate one addition step 5 uL of cell suspension in Stimulation Buffer containing 4X ULight anti cAMP can be used In this specific case 10 uL of 2X Eu cAMP tracer solution must be added in order to keep the 20 uL total assay volume e For 96 and 1536 well formats adjust volume of each assay component proportionally in order to maintain the volume ratios used for the 384 plate format e Do not mix reagents from kits with different lot numbers in order to maintain assay performance between lots 9 Instrument settings Parameter VICTOR EnVision Lamp Laser ViewLux 2X LANCE High Count Integrator Level 615 and 665 locked N A N A protocols men e ns gr _ 2 665 Pre 205 APC 665 Genio 671 8 ee Delay Time sous ET and Binning Lamp 100 100 us 100 us Lamp 662 462 or 412 Lamp 2000 us Measurement time of 20 seconds recommended for the ViewLux instrument If signal too low with 100 us 10 Typical LANCE Ultra cAMP standard curves E 35 000 E 1 500 m EnVision Laser e e ViewLux 30 0005 El 8 v EnVision Lamp 1 250 25 0004 m VICTOR 1 000 S 20 000 S 750 3 I in 15 000 D oo
8. ould be 1 000 cells well Note however that at 500 cells per well the assay window is already acceptable and therefore the optimal cell density will ultimately depend on your assay needs Additional assay development guidelines are available on PerkinElmer s website www perkinelmer com E 25 000 u E 25 000 Cells well S B 20 0004 2 CO ICi CAMP _ L 12 20 0004 500 18 e e 100 32 E 15 000 E 15 000 2 000 35 E Y 3000 36 10 000 H 10 000 FF E 5 000 W 3 000 5 7 a 3 g En NEE A lCoo cAMP J A er 0 t T T T coc 0 11 10 9 8 7 6 5 F 0 Log CAMP M Log Forskolin M Figure 2 Determination of optimal cell density Left panel cAMP standard curve right panel cell and forskolin cross titration 7 Reagent preparation 7 1 Stimulation Buffer The recommended Stimulation Buffer for cell based assays is 1X HBSS 5 mM HEPES 0 5 mM IBMX 0 1 BSA pH 7 4 Make fresh To prepare 15 mL of Stimulation Buffer add the following to a tube 14 mL of 1X HBSS Invitrogen cat 14025 092 75 Lof 1M HEPES Invitrogen cat 15630 080 30 uL of 250 mM IBMX dissolved in DMSO Sigma cat 17018 200 uL of 7 5 BSA Stabilizer included in the kit Adjust pH to 7 4 with 0 1N NaOH and complete volume to 15 mL with 1X HBSS Alternative buffers such as cell culture medium containing 10 FBS and phenol red can also be used For cAMP standard curves addition of 0 5 mM
9. ple 5 Reagents not supplied in the kit Item Recommended source Product no no phenol red Foren YS re sem mo OptiPlate 384 white PerkinElmer 6007290 pack of 50 6007299 pack of 200 ProxiPlate 384 Plus white PerkinElmer 6008280 pack of 50 E AN EAN OptiPlate 96 white PerkinElmer 6005290 pack of 50 A raci n OptiPlate 1536 white PerkinElmer 6004290 pack of 50 IN A AN 6 Assay optimization guidelines The following protocol assumes that both the cell number and stimulation conditions have been optimized as these parameters often vary for each receptor and cell line It is therefore strongly recommended to generate either forskolin Gs and Gi receptors or full agonist Gs receptors concentration response curves in order to determine the optimal cell number per well We suggest testing from 250 to 5 000 cells per well in a 20 uL assay The optimal cell number will be the one for which the forskolin or agonist concentration response curve covers most of the dynamic range of the cAMP standard curve IC o ICao This typically corresponds to the cell density giving the highest signal to background S B ratio calculated using the maximal signal untreated cells and the minimal signal obtained with a saturating concentration of agonist or forskolin fully activated cells From the example presented below Figure 2 the optimal cell concentration selected for subsequent experiments ex agonist dose response curves w
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