Assay ID Assay name Description Components of the assay SYS-A124 ADRB2/ARRB2 targetscreener

Transcription

1 ADRB2/ARRB2 targetscreener Assay SYS-A124 SYS-A124C5 systasy bioscience GmbH Adams-Lehmann-Str München Tel. +49 (0) Fax. +49 (0) Product Description The ADRB2/ARRB2 targetscreener Assay has been designed to monitor human ADRB2 receptor activation using the luc2 firefly luciferase gene (Fluc) as final readout. The targetscreener Assay components are to be transfected into U2OS cells, which are stimulated with agonists and/or antagonists, followed by luciferase readings. For a control agonist and antagonist, the addition of isoproterenol and the adrenergic receptor inhibitor propranolol is recommended. Assay ID Assay name Description Components of the assay SYS-A124 ADRB2/ARRB2 targetscreener Technology Principle Monitor ADRB2 receptor activation using a firefly luciferase readout Plasmids encoding 1. SYS-V357, padrb2-v2r-ntev-tevs-gv 2. SYS-V586, parrb2-ctev 3. SYS-V747, pg10-fluc Cells 1. SYS-C5, U2OS (only SYS-A124C5) The ADRB2/ARRB2 targetscreener Assay allows the monitoring of ADRB2 receptor activation. The assay is based on the splitsensor technology, which is a highly sensitive, flexible and easy-to-use reporter assay system to robustly and quantitatively monitor the activity of receptor tyrosine kinases (RTKs) under diverse stimuli conditions (e.g. compound concentrations, time points etc.). The technology is based on the functional complementation of TEV protease fragments fused to interaction partners of choice. Addition of an agonist, such as isoproterenol activates the GPCR ADRB2, resulting in the recruitment of the adapter-arrestin (ARRB2). In this setup, the GPCR is fused to the V2R C-terminal domain (not shown), the NTEV fragment, a TEV protease cleavage site (tevs) and the artificial transcriptional co-activator GAL4-VP16 (GV). ARRB2 is fused to the CTEV fragment. An activation-dependent interaction between the GPCR and ARRB2 causes the NTEV and CTEV fragments of the TEV protease to reconstitute its proteolytic activity. Next, GV is released and translocates to the nucleus where it binds to upstream activating sequences (10xUAS) to initiate transcription of the firefly (Fluc) reporter gene. In turn, Fluc catalyzes the reaction of the substrate luciferin to oxyluciferase and light, enabling a quantitative readout. A Renilla luciferase (Rluc) driven by the thymidine kinase (TK) promoter (not provided) may be co-transfected as internal control. Figure 1 Schematic representation of the GPCR/ARRB2 assay. ADRB2/ARRB2 targetscreener Assay, SYS-A124 SYS-A124C5 1

2 Product Information Target: Adapter: Ligand: Inhibitor: Specification: Species: Format: Storage: Assay medium: Maintenance medium: ADRB2 ARRB2 Isoproterenol Propranolol ADRB2 receptor activity can be measured using a firefly luciferase readout. Human 5 vials of plasmid DNA, 2 vials of U2OS cells (only SYS-A124C5) DNA: at -20 C; cells at - 80 C up to 2 weeks or at -196 C for more than 2 weeks in McCoy s 5A with GlutaMAX, 0.5 % dialyzed FBS in McCoy s 5A with GlutaMAX, 10% FBS Validation of Performance Performance: Dose-response curve for the ADRB2 agonist isoproterenol. Figure 2. Dose response curve of ADRB2 activation using isoproterenol. The EC 50 is at a concentration of 0.945µM isoproterenol. Assay performed in U2OS cells, error bars represent s.e.m. Dose-response curve for the ADRB2 antagonist asenapine. Figure 3. Dose response curve of ADRB2 inactivation using propranolol. The IC 50 is at a concentration of 4.98nM propranolol. Assay performed in U2OS cells with a constant epinephrine stimulus (1µM), error bars represent s.e.m. ADRB2/ARRB2 targetscreener Assay, SYS-A124 SYS-A124C5 2

3 Additionally Required Material Media Supplier Cat. Number McCoy s 5A with GlutaMAX, 500 ml ThermoFisher Opti-MEM ThermoFisher Serum and other additives Fetal Bovine Serum (Heat Inactivated), ThermoFisher ml (EU approved) Dialyzed FBS, 100ml ThermoFisher Nonessential amino acids (NEAA), 100, ThermoFisher ml GlutaMAX, 100x, 100ml ThermoFisher Penicillin-Streptomycin Mixture Lonza DE17-602E Transfection reagent Lipofectamine 2000 ThermoFisher Suggested material for 96-well plates and coating 96-well white plates Falcon Poly-L-Lysine hydrobromide, mw , 100mg Sigma-Aldrich P MG Suggested assay control reagents Isoproterenol hydrochloride Sigma-Aldrich I6504 Epinephrine hydrochloride Sigma-Aldrich E4642 Propranolol Sigma-Aldrich P0884 Suggested material for luciferase assay 5x Passive Lysis Buffer Promega E1941 Steady-Glo Luciferase Assay System Promega E2520 If using Renilla as control: Dual- Luciferase Reporter 1000 Assay System Promega E1980 Handling of Cells Coating of 96-well plates with Poly-L-Lysine (PLL): 1. Make a PLL solution of 0.02 mg/ml diluted in H2O. 2. Cover each well in a 96-well plate with PLL solution and incubate for min. 3. Wash wells 2 times with H2O and let plates air-dry. 4. Optional: irradiate the plates with UV-light for 30 min. ADRB2/ARRB2 targetscreener Assay, SYS-A124 SYS-A124C5 3

4 Thawing cells: 1. Thaw the cells by placing the cryo vial directly from liquid nitrogen or dry ice into a 37 C water bath (~ 2 min). 2. Add 1 ml 37 C warm Maintenance medium and mix medium with the cell suspension by gentle pipetting. 3. Centrifuge for 4 min at 800 rpm. 4. Carefully remove the supernatant and resuspend the cell pellet in 12 ml Maintenance medium by pipetting and plate the cells onto a 10 cm dish. Propagating and cultivating cells: 1. PLL-coat 10cm or 15cm cell culture dishes. 2. Prewarm Maintenance medium, PBS and Trypsin/EDTA in a 37 C water bath. 3. Place your cells under the cell culture hood. Remove and discard medium. 4. Briefly rinse the cells with PBS and then add as much as Trypsin/EDTA that the whole surface of the dish is covered (e.g. 6 ml on a 15-cm dish, 3 ml on a 10-cm dish) 5. Place the dish for 2-5 minutes in the incubator and let the cells detach. Observe cells under a microscope, until they are dispersed. 6. Add the same volume of medium (containing FBS) as used for Trypsin/EDTA to stop the reaction. Aspirate the cells by gently pipetting. 7. Transfer the mixture to a 15 ml Falcon and centrifuge at 500 g for 5 minutes. 8. Aspirate the supernatant and resuspend the cells in about 5 ml fresh medium. 9. Depending on the estimated days of propagation split the cells 1:3-1: Add fresh medium to the cells. Medium to be Prepared Maintenance medium: Assay medium: McCoy s 5A with GlutaMAX + 10% heat-inactivated FBS + 1% Pen/Strep McCoy s 5A with GlutaMAX + 0.5% heat-inactivated FBS, dialyzed + 1% nonessential amino acids (NEAA) (1mM) ADRB2/ARRB2 targetscreener Assay, SYS-A124 SYS-A124C5 4

5 Protocol for the targetscreener Assay On-plate transfection of targetscreener Assay components 1. One day before the assay, plate the amount of cells needed for the assay: rinse (with PBS), trypsinize and detach, spin down, and resuspend the cells in Maintenance medium (in 100µl per 96-well). Plate cells on PLL-coated 96-well plates. 2. Mix the plasmid DNAs according to the scheme below. 3. Mix Opti-MEM, GlutaMAX and LF Vortex. 5. Incubate 2 minutes. 6. Combine LF2000/Opti-MEM and DNA mix. 7. Vortex. 8. Incubate 20 minutes at room temperature. 9. Remove the medium from the wells without aspirating cells. 10. Add 30µl/96-well of the DNA/LF2000/Opti-MEM mix onto the cells. 11. Incubate 2h at 37 C. 12. Add 60µl/96-well of Maintenance medium. 13. Incubate 16-24h for protein expression. 14. Exchange the Maintenance medium with 60 µl Assay medium. 15. Incubate 16-18h to induce starvation. 16. Add compounds in a volume of 60µl/96-well (added as a 2x concentrated mix diluted in Assay medium). 17. Incubate 1h. Optional for stimulation with agonists only: Incubate for 6h and proceed directly to step Add the isoproterenol positive stimulus in a volume of 12µl/96-well (added as a 10x concentrated mix diluted in Assay medium). 19. Incubate 6h. 20. Run luciferase assay (see below). Luciferase Assay protocol 1. Remove the medium and add 25µl of 1x Passive Lysis buffer to each well. 2. Incubate the cell lysates for 10min on a rocking platform at room temperature. 3. Perform luciferase readout: Add 50 µl of firefly substrate (from commercial Steady-Glo Luciferase Kit, Promega), read your plate using a luciferase plate reader. OR: Perform the Dual luciferase readout if using Renilla as control: a. Add 50 µl of firefly substrate (from commercial Dual Luciferase Kit, Promega), read your plate using a luciferase plate reader. b. Add 50 µl of Renilla substrate (from commercial Dual Luciferase Kit, Promega, read your plate using a luciferase plate reader. ADRB2/ARRB2 targetscreener Assay, SYS-A124 SYS-A124C5 5

6 Table 1: Amounts of plasmids, medium, and cells used per 96-well plate DNA/Single well DNA/Per plate Unit ID of plasmid, name µg SYS-V357, padrb2-v2r-ntev-tevs-gv µg SYS- V586, parrb2-ctev µg SYS-V747, pg10-fluc Cells/96-well Cells/per plate Cell type U2OS µl/96-well Volume/per plate Unit Medium/reagent µl Lipofectamine µl GlutaMAX 30 3 ml Opti-MEM 60 6 ml Maintenance medium, for step ml Assay medium, for step ml Compound in Assay medium, for step ,2 ml Isoproterenol in Assay medium, for step 18 ADRB2/ARRB2 targetscreener Assay, SYS-A124 SYS-A124C5 6