Cell Line Descriptions T-Rex™ Tango™ P2RY14-Bla U2OS Cells Contain

Version No.: Tango™ Validation Packet Page 1 of 3 01Sep08

Optimization of the T-REx™ Tango™ P2RY14-bla U2OS Cell Line

T-REx™ Tango™ P2RY14-bla U2OS cells

Catalog Numbers – K1858

Cell Line Descriptions

T-REx™ Tango™ P2RY14-bla U2OS cells contain the human Purinergic Receptor P2Y14 (P2RY14) linked to a TEV protease site and a Gal4-VP16 transcription factor stably integrated into the Tango™ GPCR-bla U2OS parental cell line. This parental cell line stably expresses a beta-arrestin/TEV protease fusion protein and the beta-lactamase reporter gene under the control of a UAS response element.

The T-REx™ Tango™ P2RY14-bla U2OS cells have been functionally validated for Z’ factor and EC 50 concentrations of Uridine 5'-diphosphoglucose disodium salt (UDP-glucose) (Figure 1). In addition, T-REx™ Tango™ P2RY14-bla U2OS 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 T-REx™ Tango™ P2RY14-bla U2OS Cell Line Page 2 of 3

Validation Summary Primary Agonist Dose Response

bla Testing and validation of this assay was evaluated Figure 1 — T-REx™ Tango™ P2RY14- U2OS cells dose response to Uridine 5'-diphosphoglucose disodium salt in a 384-well format using LiveBLAzer™-FRET B/G (UDP-glucose) under optimized conditions Substrate.

T-REx Tango P2RY14 1. Uridine 5'-diphosphoglucose disodium 8 salt (UDP-glucose) dose response Dividing 7 under optimized conditions Cryo 6 Dividing Cells 5 EC 50 1.8 µM 4 Z’-factor 0.72 3 Recommended cell no. /well = 10,000 2 Response Ratio Recommended Stim. Time = 5 hrs 1 Max. [Stimulation] = 100 µM 0 -9 -8 -7 -6 -5 -4 -3 2. Alternate agonist dose response Log [M] UDP-glucose

Uridine 5’-diphosphoglucuronic acid T-REx™ Tango™ P2RY14-bla U2OS cells (10,000 cells/well) trisodium salt (UDPGA) EC 50 were plated in a 384-well format and incubated for 16-20 = 40.5 µM hours. Cells were stimulated with a dilution series of Uridine 5'-diphosphoglucose disodium salt (UDP-glucose) (Sigma 3. Antagonist dose response U4625) 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 were no known antagonist obtained using a standard fluorescence plate reader and Response Ratios plotted for each replicate against the concentrations of Uridine 5'-diphosphoglucose disodium salt (UDP-glucose).

Alternate Agonist Dose Response

Figure 2 — T-REx™ Tango™ P2RY14-bla U2OS cells dose response to UDP-glucose and UDPGA. 9 UDPGA 8 7 UDPG 6 5 4 3 2 Response Ratio 1 0 -9 -8 -7 -6 -5 -4 -3 -2 Log [M] compound

T-REx™ Tango™ P2RY14-bla U2OS cells (10,000 cells/well) were plated in a 384-well format and incubated for 16-20 hours prior to stimulation with UDP-glucose (Sigma U4625) and UDPGA (Sigma U6751) 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 Response Ratios plotted against the indicated concentrations of agonist.

References

1) Chambers, Jon K. et. al. (2000) A G protein-coupled receptor for UDP-glucose . J. Biol. Chem. , 275 , 10767-10771.

2) Byeong-Chel, Lee et. al. (2003) P2Y-like receptor, GPR105 (P2Y14), identifies and mediates chemotaxis of bone-marrow hematopoietic stem cells . Genes and Development , 17 , 1592-1604.

3) Skelton, Lisa et. al. (2003) Human immature monocyte-derived dendritic cells express the G protein-coupled receptor GPR105 (KIAA0001, P2Y14) and increase intracellular calcium in response to its agonist, Uridine diphosphoglucose . J. Immunol. , 171 , 1941-1949.

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]