Version No.: GeneBLAzer ® Validation Packet Page 1 of 2 01Sep08 Optimization of the GeneBLAzer® CALCRL:RAMP3-CRE-bla FreeStyle™ 293F Cell Line GeneBLAzer ® CALCRL:RAMP3-CRE-bla FreeStyle™ 293F Cells Catalog Numbers – K1439 Cell Line Descriptions GeneBLAzer ® CALCRL:RAMP3-CRE-bla FreeStyle™ 293F cells contain the human Adrenomedullin Receptor 2 (CALCRL:RAMP3) (Accession # NM_005795.3 NM_005856.1) stably integrated into the CellSensor ® CRE-bla Freestyle™ 293F cell line. CellSensor ® CRE-bla Freestyle™ 293F cells (Cat. no. K1636) contain a beta-lactamase (bla ) reporter gene under control of the cAMP response element (CRE). GeneBLAzer ® CALCRL:RAMP3-CRE-bla FreeStyle™ 293F cells are functionally validated for Z’- ® factor and EC 50 concentrations of Adrenomedullin(1-52) (Figure 1). In addition, GeneBLAzer CALCRL:RAMP3-CRE-bla FreeStyle™ 293F cells have been tested for assay performance under variable conditions. Have a question? Contact our Technical Support Team NA: 800-955-6288 or INTL: 760-603-7200 Select option 3, ext. 40266 Email: [email protected] Optimization of the GeneBLAzer® CALCRL:RAMP3-CRE-bla FreeStyle™ Page 2 of 2 293F Cell Line Validation Summary 2nd Messenger Dose Response ® bla Testing and validation of this assay was evaluated Figure 2 — GeneBLAzer CALCRL:RAMP3-CRE- FreeStyle™ 293F 2 nd messenger dose response to in a 384-well format using LiveBLAzer™-FRET B/G Adrenomedullin(1-52) under optimized conditions. Substrate. 120 110 1. Adrenomedullin(1-52) dose response 100 90 under optimized conditions 80 70 60 Dividing Cells 50 EC 50 = 506 pM 40 30 Z’-factor = 0.81 % Activation 20 10 Recommended cell no. /well = 10,000 0 -10 Recommended Stim. Time = 5 hrs -13 -12 -11 -10 -9 -8 -7 -6 Max. [Stimulation] = 100 nM log M [AM(1-52)] ® bla 2. Antagonist dose response GeneBLAzer CALCRL:RAMP3-CRE- FreeStyle™ 293F cells were tested for a response to Adrenomedullin(1-52) with a TR- FRET cAMP kit. AM(22-52) = N/A nd 3. Assay performance in 2 messenger assay. AM(1-52) = 632 pM Primary Agonist Dose Response Antagonist Dose Response ® Figure 1 — GeneBLAzer ® CALCRL:RAMP3-CRE-bla Figure 3 — GeneBLAzer CALCRL:RAMP3-CRE-bla FreeStyle™ 293F cells dose response to FreeStyle™ 293F dose response to AM(22-52) Adrenomedullin(1-52) under optimized conditions 6.5 100 6.0 90 5.5 80 5.0 70 4.5 4.0 60 3.5 50 3.0 40 2.5 30 2.0 % Inhibition 20 1.5 Response Ratio Response 10 1.0 0.5 0 0.0 -10 -12 -11 -10 -9 -8 -7 -6 -10 -9 -8 -7 -6 -5 Log M [AM(1-52)] Log [AM(22-52)] M ® GeneBLAzer ® CALCRL:RAMP3-CRE-bla FreeStyle™ 293F cells GeneBLAzer CALCRL:RAMP3-CRE-bla FreeStyle™ 293F cells (10,000 cells/well) were plated in a 384-well format and (10,000 cells/well) were plated in a 384-well format and incubated for 16-20 hours. Cells were stimulated with a incubated for 16-20 hours. Cells were exposed to AM(22-52) dilution series of Adrenomedullin(1-52) (Sigma A2327) in the (Sigma A3832) for 30 min. and then stimulated with an EC80 presence of 0.1% DMSO for 5 hours. Cells were then loaded concentration of Adrenomedullin(1-52) (Sigma A2327) in the with LiveBLAzer™-FRET B/G Substrate for 2 hours. presence of 0.1% DMSO for 5 hours. Cells were then loaded Fluorescence emission values at 460 nm and 530 nm were with LiveBLAzer™-FRET B/G Substrate for 2 hours. obtained using a standard fluorescence plate reader and % Fluorescence emission values at 460 nm and 530 nm for the Activation plotted for each replicate against the concentrations various substrate loading times were obtained using a of Adrenomedullin(1-52). standard fluorescence plate reader and the % Inhibition plotted against the indicated concentrations of AM(22-52). Have a question? Contact our Technical Support Team NA: 800-955-6288 or INTL: 760-603-7200 Select option 3, ext. 40266 Email: [email protected] .