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Ca++-Induced G Protein Coupled Receptor (GPCR) Activation Monitored via Aequorin Luminescence in the LMax Microplate Luminometer (MaxLine Application Note #42)

ith a constant agonist concentration in the injector reservoir, theLmax microplate luminometer could potentially be used to run high-thoughputluminescent assays for GPCR antagonist screening.


PRINCIPLE OF ASSAY
CHO cells expressing the apo-aequorin protein were tested in the L-max using uridine triphosphate (UTP) to activate the endogenous P2Y purinergic receptor. This receptor is known to be coupled to the Gq pathway, causing an increase in intracellular calcium. In order to form the aequorin complex, the cells were first loaded with the coenzyme coelenterazine. There are many different isoforms of this coenzyme available, for these studies. We chose to use the native coelenterazine and the coelenterazine(h) isoform.


MATERIALS
1. Lmax microplate luminometer with SOFTmax PRO for Lmax (Molecular Devices Corp.)

2. CHOMQ6 cell line stably expressing apo-aequorin, kindly provided by Jenny Stables (GlaxoWellcome Research, U.K.)

3. Coelenterazine, both native and h iso forms from Molecular Probes, Inc.; http://www.probes.com

4. UTP, Calbiochem Cat. No. 6701; Tel: 1-800-492-1110

5. Hams F-12 Cat. No. 9058, Fetal Bovine Serum Cat. No. 3000, Glutamine Pen-Strep Solution Cat. No. 9316, G418 sulfate #98529, and 1 M HEPES buffer solution Cat. # 9319 were obtained from Irvine Scientific Tel. 1-800-437-5706.

6. 10X Hanks Balanced Salt Solution, GIBCO BRL Cat# 14065-056; Tel 800-8286686

7. Clear-bottomed white 96-well microplates. CorningCostar Cat. No. 3903. Tel: 18004921110.

8. The native coelenterazine and h isoform loading solutions (2.5 M) were prepared by dilution in Hanks Balanced salt solution + 20 mM HEPES (H&H) with 1 mg/ml BSA.


PREPARATION OF CELLS
1. The stable CHOMQ6 cell line expressing apo-aequorin was maintained in F12 growth
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The Light Diagnostics Universal Amplicon Detection Assay (ADA) is intended for use in the qualitative detection and quantitation of DNA generated by in vitro nucleic acid amplification. For research
{3,8-diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphenanthridinium diiodide}. ,250 ug/ml Propidium Iodide (PI) in PBS.
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