A2B Human Adenosine GPCR Cell Based Agonist & Antagonist cAMP LeadHunter Assay - TW

Procedure Summary:

Human recombinant adenosine A2B receptors stably expressed in CHO-X13 cells are used. Test compound and/or vehicle is incubated with the cells (1 x 10^5/ml) in modified HBSS pH 7.4 buffer for 30 minutes at 37°C. The reaction is evaluated for cAMP levels by TR-FRET. Test compound-induced cAMP increase by 50 percent or more (≥50%) relative to the 10 μM NECA control response indicates possible adenosine A2B receptor agonist activity. Test compound-induced inhibition of the NECA (1 μM) induced cAMP response by 50 percent or more (≥50%) indicates receptor antagonist activity. These same concentrations are concurrently applied to a separate group of treated cells and evaluated for possible compound-induced cytotoxicity only if significant inhibition of cAMP release is observed (Cat. #301100).

Incubation:

30 min at 37°C

Control Inhibitor:

8-Cyclopentyl-1,3-dipropylxanthine

Control Activator:

NECA

Criteria For Significance:

≥ 50% Increase in cAMP relative to NECA response, ≥ 50% Inhibition of NECA-induced cAMP increase

Test Concentration / Dose:

Compounds are screened at 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and 0.003 μM

Test Sample Requirements:

75 μl of 10 mM stock or 1 mg (pre-weighed) powder (assuming FW ≦500 and purity 100%)

Minimum Order Quantity:

16