Version No.: GeneBLAzer ® Validation Packet Page 1 of 2 01Sep08

Optimization of the GeneBLAzer® CALCRL:RAMP2-CRE-bla FreeStyle™ 293F Cell Line

GeneBLAzer ® CALCRL:RAMP2-CRE-bla FreeStyle™ 293F Cells

Catalog Numbers – K1438

Cell Line Descriptions

GeneBLAzer ® CALCRL:RAMP2-CRE-bla FreeStyle™ 293F cells contain the human Receptor 1 (CALCRL:RAMP2), (Accession #’s NM_005795.3, BC02795.1) 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 under control of the cAMP response element (CRE).

GeneBLAzer ® CALCRL:RAMP2-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:RAMP2-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:RAMP2-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:RAMP2-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 under optimized conditions 90 80 70 Dividing Cells 60 EC = 920 pM 50 50 40

Z’-factor = 0.62 % Activation 30 20 Recommended cell no. /well = 10,000 10 0 Recommended Stim. Time = 5 hrs -13 -12 -11 -10 -9 -8 -7 -6 Max. [Stimulation] = 300 nM log M [AM(1-52)]

® 2. Assay performance in 2 nd messenger GeneBLAzer CALCRL:RAMP2-CRE-bla FreeStyle™ 293F cells were tested for a response to Adrenomedullin(1-52) with a TR- assay. FRET cAMP .

AM(1-52) =2.6 nM

3. Antagonist dose response

AM(22-52) = 72 nM

Primary Agonist Dose Response Antagonist Dose Response

Figure 1 — GeneBLAzer ® CALCRL:RAMP2-CRE-bla Figure 3 — GeneBLAzer ® CALCRL:RAMP2-CRE-bla FreeStyle™ 293F cells dose response to FreeStyle™ 293F dose response to AM(22-52) Adrenomedullin(1-52) under optimized conditions

4.5 110 100 4.0 90 3.5 80 3.0 70 60 2.5 50 2.0 40 1.5 30 % Inhibition 20

Response Ratio Response 1.0 10 0.5 0 0.0 -10 -12 -11 -10 -9 -8 -7 -6 -10 -9 -8 -7 -6 -5 -4 Log [AM1-52] M Log [AM-(22-52)] M

GeneBLAzer ® CALCRL:RAMP2-CRE-bla FreeStyle™ 293F cells GeneBLAzer® CALCRL:RAMP2-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 the Fluorescence emission values at 460 nm and 530 nm for the response ratio was plotted for each replicate against the various substrate loading times were obtained using a concentrations 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]