Optimization of the Geneblazer® CALCRL:RAMP1-CRE-Bla Freestyle™ 293F Cell Line

Version No.: GeneBLAzer® Validation Packet Page 1 of 3 01Sep08

Optimization of the GeneBLAzer® CALCRL:RAMP1-CRE-bla FreeStyle™ 293F Cell Line

GeneBLAzer® CALCRL:RAMP1-CRE-bla FreeStyle™ 293F Cells

Catalog Numbers – K1437

Cell Line Descriptions

GeneBLAzer® CALCRL:RAMP1-CRE-bla FreeStyle™ 293F cells contain the human Calcitonin Gene Related Peptide Receptor (CALCRL:RAMP1), (Accession # NM_005795.3 NM_005855.2) 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:RAMP1-CRE-bla FreeStyle™ 293F cells are functionally validated for Z’- ® factor and EC50 concentrations of Alpha-CGRP (Figure 1). In addition, GeneBLAzer CALCRL:RAMP1-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:RAMP1-CRE-bla FreeStyle™ Page 2 of 3 293F Cell Line

Validation Summary Primary Agonist Dose Response

® Testing and validation of this assay was evaluated Figure 1 — GeneBLAzer CALCRL:RAMP1-CRE-bla FreeStyle™ 293F cells dose response to Alpha-CGRP in a 384-well format using LiveBLAzer™-FRET B/G under optimized conditions Substrate. 5

1. Alpha-CGRP dose response under 4 optimized conditions 3 EC50 = 135 pM Z’-factor = 0.80 2

Recommended cell no. /well = 10,000 Response Ratio 1 Recommended Stim. Time = 5 hrs 0 Max. [Stimulation] = 100 nM -13 -12 -11 -10 -9 -8 -7 Log [αCGRP] M 2. Alternate agonist dose response ® GeneBLAzer CALCRL:RAMP1-CRE-bla FreeStyle™ 293F cells (10,000 cells/well) were plated in a 384-well format and Beta-CGRP = 118 pM incubated for 16-20 hours. Cells were stimulated with a dilution series of Alpha-CGRP (Sigma C0167) in the presence 3. Antagonist dose response of 0.1% DMSO for 5 hours. Cells were then loaded with LiveBLAzer™-FRET B/G Substrate for 2 hours. Fluorescence Alpha-CGRP(8-37) = 112 nM emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader and % Activation plotted for each replicate against the concentrations of Alpha-CGRP.

Alternate Agonist Dose Response

Figure 2 — GeneBLAzer® CALCRL:RAMP1-CRE-bla Assay Testing Summary FreeStyle™ 293F dose response to alpha-CGRP and beta- CGRP

120 nd 110 4. Assay performance in 2 messenger 100 90 assay. 80 70 60 Alpha-CGRP = 64 pM 50 alpha-CGRP 40 30 beta-CGRP % Activation 20 10 0 -10 -13 -12 -11 -10 -9 -8 -7 Log [Agonist] M

GeneBLAzer® CALCRL:RAMP1-CRE-bla FreeStyle™ 293F cells (10,000 cells/well) were plated in a 384-well format and incubated for 16-20 hours prior to stimulation with Alpha-CGRP (Sigma C0167), Beta-CGRP (Sigma C1044) over the indicated concentration range in the presence of 0.1% DMSO for 5 hours. Cells were then loaded with LiveBLAzer™-FRET B/G Substrate for 2 hours. Emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader and the % Activation plotted against the indicated concentrations of agonist. The data shows the correct rank order potency.

Antagonist Dose Response 2nd Messenger Dose Response

Figure 3 — GeneBLAzer® CALCRL:RAMP1-CRE-bla Figure 4 — GeneBLAzer® CALCRL:RAMP1-CRE-bla FreeStyle™ 293F dose response to alpha-CGRP(8-37) FreeStyle™ 293F 2nd messenger dose response to Alpha- CGRP under optimized conditions.

110 120 100 110 90 100 80 90 70 80 60 70 60 50 50 40 40 30 30 % Inhibition 20 % Activation 20 10 10 0 0 -10 -10 -10 -9 -8 -7 -6 -5 -4 -13 -12 -11 -10 -9 -8 -7 -6 Log [aCGRP(8-37)] M Log M [alpha-CGRP]

GeneBLAzer® CALCRL:RAMP1 CRE-bla FreeStyle™ 293F cells GeneBLAzer® CALCRL:RAMP1-CRE-bla FreeStyle™ 293F cells (10,000 cells/well) were plated in a 384-well format and were tested for a response to Alpha-CGRP with a TR-FRET incubated for 16-20 hours. Cells were exposed to alpha- cAMP kit. CGRP(8-37) (Sigma C2806) for 30 min. and then stimulated with an EC80 concentration of Alpha-CGRP (Sigma C0167) in the presence of 0.1% DMSO for 5 hours. Cells were then loaded with LiveBLAzer™-FRET B/G Substrate for 2 hours. Fluorescence emission values at 460 nm and 530 nm for the various substrate loading times were obtained using a standard fluorescence plate reader and the % Inhibition plotted against the indicated concentrations of alpha-CGRP(8-37).

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]