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Screening for potential beta 2-adrenergic receptor antagonists using CypHer5E and IN Cell Analyzer 1000

for potential ligands of non-G-protein coupled receptors (5) and orphan receptors.


Materials

Other materials required
LOPAC compound library (Sigma)

HEK293 cell line expressing VSV-G epitope tagged β2-adrenergic receptor

Culture medium:
MEM (Sigma)
10% (v/v) FBS (Sigma)
200 µg/ml Geneticin 418 (Sigma)
L-glutamine (Gibco-BRL)
Non-essential amino acids (Sigma)

Assay medium: MEM, phenol red free (Invitrogen)

Agonist: isoproterenol (Sigma)

Antagonist: alprenolol (Sigma)

Hoechst™ 33342 (Molecular Probes)

Poly-D-lysine, 5 mg (Sigma)

96-well assay plate (Greiner Bio-One Ltd.)


Protocol
LOPAC compounds and plate configuration
1 LOPAC’s 640 pharmacologically active compounds were organized by receptor class/activity into eight 96-well plates (80 compounds per plate at 1 mM) as follows:

Plate 1: adenosines/purinergics

Plate 2: adrenergics/histaminergics

Plate 3: cholinergics/ion channel modulators

Plate 4: dopaminergics

Plate 5: glutaminergics

Plate 6: signal transduction agents/opioids

Plate 7: serotonergics

Plate 8: enzyme inhibitors/GABAergics

The compounds were further diluted in assay medium to give final well concentrations of 100 nM. This value was determined during a pre-screen library titration study to be the optimum screen concentration for the LOPAC compounds in 0.01% DMSO. Agonist and antagonist controls were placed symmetrically in columns 1 and 12 of each plate. Twelve replicate wells contained a known agonist, isoproterenol, and four replicate wells contai
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