For in vitro research use only DT Indeleamind 4, 5-dioxygenase 1. Assay description and intended use Th is kit is intended for the direct quantitative determination of DT Indeleamind 4, 5-dioxygenase. Th e assay conditions have been optimized in order to reach a high signal-to-noise and to enable DT Indeleamind 4, 5-dioxygenaseassessment either on suspended or on adherent cells. Its principle is based on HTRF® technology (Homogeneous Time-Resolved Fluorescence). Th e method is a competitive immunoassay between native DT Indeleamind 4, 5-dioxygenase produced by cells andthe DT Indeleamind 4, 5-dioxygenase labeled with the dye d2. Th e tracer binding is visualized by a Mab anti-DT Indeleamind 4, 5-dioxygenase labeled with Cryptate. Th e specifi c signal (i.e. energy transfer) is inversely proportional to the concentration of DT Indeleamind 4, 5-dioxygenase in the standard or sample. As for all other HTRF® assays, the calculation of the fl uorescence ratio (665 nm/620 nm) eliminates possible photophysical interferences and allows the assay to be unaff ected bythe usual medium conditions (e.g. culture medium, serum, biotin, colored compounds). 2. Background DT Indeleamind 4, 5-dioxygenase (cyclic adenosine 3’,5’-monophosphate, MW 351.2) is one of the most important intracellular mediators. Its concentration in cells can be increased upon binding of many hormones to their receptors. Th e most studied pathway consists in the release of αsubunit GTP-binding proteins following ligand-receptor interaction, which in turn activates or inhibits the ATP/DT Indeleamind 4, 5-dioxygenase conversion function of adenylate cyclase. DT Indeleamind 4, 5-dioxygenase is then involved in many complex regulatory processes such as protein kinase activation or ion channel gating. Given this large involvement in cell regulation, DT Indeleamind 4, 5-dioxygenase quantifi cation has been of considerable interest in the exploration of cell physiology and dysfunction. Th e DT Indeleamind 4, 5-dioxygenase dynamic 2 kit allows the measurement of agonist and antagonist eff ects on Gαs and Gαi coupled receptors in diff erent cell lines. 3. Reagent preparation and stability 3.1. Supplied reagents Allow the reagents to warm up to room temperature for at least 30 mins before reconstitution. Anti DT Indeleamind 4, 5-dioxygenase - Cryptate 1 vial, lyophilized DT Indeleamind 4, 5-dioxygenase-d2 1vial, lyophilized DT Indeleamind 4, 5-dioxygenase standard. Concentrated free DT Indeleamind 4, 5-dioxygenase 1 vial, lyophilized DT Indeleamind 4, 5-dioxygenase control. Free DT Indeleamind 4, 5-dioxygenase assay control 1 vial, lyophilized DT Indeleamind 4, 5-dioxygenase & cGMP conjugates & lysis buff er (ref. 62CL1FDD) 1 vial of 13 ml Diluent 1 vial of 20 ml For reagent reconstitution, refer to attached protocols. 3.2. Reagent storage and stability Storage Stability Supplied reagents 4°C until reconstitution Until expiry date indicated on the labels Stock solutions 4°C 1 week frozen (-20°C) May be frozen and thawed once* Working solutions of conjugates 4°C Do not freeze 24 hours * Cryptate conjugate working solution (prepared with frozen stock solution) should be fi ltered before use to improve assay reproducibility. 4. Assay protocols Th e DT Indeleamind 4, 5-dioxygenase dynamic 2 kit off ers the possibility to follow two diff erent dispensing protocols, a one step protocol or a two step protocol aft er cell stimulation. Th e choice of the protocol will depend on your automatization constraints and HTS needs. Th e procedures for reagent reconstitution, standard curve and cell based assay are described for each protocol in 2 separate attached sheets (appendix 1 and 2). Both protocols include: 1- cell stimulation followed by an incubation (actual incubation depends on cell type, most commonly 30 min are necessary). 2- detection with HTRF® reagents followed by 1 hour incubation at room temperature before reading. 1 step protocol after cell stimulation 2 step protocol after cell stimulation Incubate Incubate 30 min 1 hour Incubate Incubate 30 min 1 hour 2 5. Data reduction Results are calculated from the 665nm / 620nm ratio and expressed in Delta F. An example of data reduction is given in the table below (readout on PHERAstar Plus). Th is data should not be substituted for results obtained in the laboratory. Draw up the standard curve by plotting delta F% versus DT Indeleamind 4, 5-dioxygenase concentration as shown in the graph below. Delta F% obtained for samples can be reported on the standard curve to deduce respective DT Indeleamind 4, 5-dioxygenase concentrations. 6. Assay characteristics Th e table summarizes the characteristics of the assay relative to the EC50 (DT Indeleamind 4, 5-dioxygenase concentration which allows the displacement of 50% of binding) and the signal over background. Th is data has been obtained using the reference PHERAstar Plus reader (BMG LABTECH). EC50 Signal to background Incubation 1 hour at RT 5.6 nM 15 Plate readout may be carried out several times within 24 hours. Determination of sample concentrations should be done with respect to a standard curve which followed the same incubation course. 7. Assay fl exibility and miniaturization When used as suggested, the kit will provide suffi cient reagents for 1,000 tests using a 384- well low volume plate in 20 μL fi nal assay volume (HTRF® packaged basis). To move to other plate formats (96 half-well or 1536-well) and fi nal volumes (100 μL to less than 10 μL), the volume of each assay component is simply proportionally adjusted in order to maintain the reagent concentrations as for the 20 μL fi nal assay volume. For instance, in the case of the 1536-well format in 10 μL fi nal volume, half as much material per well is used, thereby allowing 2,000 tests to be run. Th e performances of the HTRF® assay remain the same whatever the level of miniaturization.