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Gq-coupled Receptor Assays Using FLEXstation

Sub>50 value for ADP was approximately 190 M as determined by phosphoinositide hydrolysis assay (1, Fig. 2).


MATERIALS
Characterization of cP2Y11 by Calcium Flux Assay

1. Cells: CF2Th canine thymocyte cells that stably overexpress the canine P2Y11 Gq-coupled receptor (1).

2. Reagents: FLEXstation Calcium Assay Kit (Molecular Devices, cat# R8041; Tel: 1-800-635-5577), ADP (Sigma, cat# A2754; Tel: 1-800-325-3010), probenecid (Sigma, cat# P8761).

3. Tissue culture microplates: 96-well, black-walled, clear-bottomed plates (Costar, cat# 3603; Tel: 1-800-492-1110).

4. Compound plates: 96-well, V-bottom clear polypropylene plates (E&K, cat# 651201; Tel: 408-378-2013).

5. Pipet tips: For FLEXstation: 200 L nonsterile, clear polypropylene tips (Molecular Devices, cat# 9000-0623).

6. Medium and buffers: Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum as previously described (9), trypsin-EDTA solution (Gibco/Invitrogen, cat# 25200-072; Tel: 1800-828-6686), Hank's balanced salt solution (HBSS) (10X) (Gibco, cat# 14065056; Tel: 1-800-828-6686), HEPES 1M buffer solution (Gibco, cat#15630-080).


METHODS
Cell Preparation
CF2Th cells were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum. Cells were seeded the night before the experiment at a concentration of 2.55 x 104 cells/well in a volume of 100 L per well of a 96-well microplate. Cells were incubated at 37 C in 5% CO2 overnight.

Cell Loading Procedure
1X Reagent Buffer was prepared (1X HBSS/20 mM HEPES, pH 7.4) with the reagents described above. Loading
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