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18. HEK cells were grown in DMEM, 10% (v/v) heat
inactivated fetal bovine serum, 100 units/ml penicillin-G
and 100 lg/ml streptomycin sulfate, 1 mM sodium pyru-
vate, 0.1 mg/ml hygromycin B, and 500 lg/ml G418 in a
humidified atmosphere at 37 ꢁC, 6% CO2. One day prior
to fluorescent imaging plate reader (FLIPR) functional
assay, hEP1 stably transfected HEK293 cells were seeded
at 35,000 cells/well. Agonist (PGE2) and antagonists were
prepared in DMSO at 200· concentration and stored at
À20 ꢁC. For the FLIPR assay, agonist and antagonist
drugplates were thawed and diluted 10-fold to 20 · in
Hanks Balanced Salt Solution (HBSS) containing20 mM
HEPES, pH 7.4 (HBSS/HEPES). FLIPR no wash dye
(Molecular Probes R8033) was diluted in HBSS/ HEPES.
Confluent cell monolayers were washed twice in HBSS/
HEPES buffer. HBSS/HEPES buffer and FLIPR dye were
added to cell monolayers and cells were incubated for 1 h
at 37 ꢁC in the presence of CO2 to enable dye loading. The
plates containingthe dye loaded cells, diluted antagonists
and agonist were placed in the FLIPR. Addition of
compound to cells and fluorescence readingwere per-
formed automatically by the FLIPR. Fluorescence was
monitored for 15 min (10 min antagonist preincubation
followed by 5min agonist stimulation). For each antago-
nist tested, a dose response curve (from 0.001 to 30 lM)
was first generated for PGE2. Three additional dose
response curves were generated in which cells were
preincubated in the presence of fixed concentrations of
antagonist for 10 min followed by challenge with increas-
ingconcentrations of PGE .
2