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27. The CRTH2 or DP radioligand binding assay was performed on 293 cells stably
expressing human CRTH2or DP, respectively. To measure binding, [3H]-PGD2
was incubated together with 293(hCRTH2) cells in the presence of increasing
concentrations of compounds. After washing, the amount of [3H]-PGD2 that
remained bound to the cells was measured by scintillation counting and the
concentration of compounds required to achieve a 50% inhibition of [3H]-PGD2
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Vacutainer tubes (VWR). IBMX (3-Isobutyl-1-methylxanthine, Sigma) was
added to whole blood at a final concentration of 1 mM. 200
treated blood was incubated in the presence of a small molecule DP antagonist
in a 96 well plate at 37 °C for 30 min. 20 L of the antagonist incubated blood
lL/well of IBMX
l
was then aliquoted into a 96 well plate containing PGD2 (Cayman Chemicals)
at various concentrations. The plate was incubated at 37 °C for 60 min. After
the PGD2 stimulation, the blood was lysed with 55 lL/well of Tropix Lysis
buffer (ABI) and subsequently assayed for cAMP levels using the cAMP-Screen
DirectÒ Immunoassay System (ABI). The PGD2 stimulated cAMP curves were fit
Genedata Screener Condoseo (Genedata AG) using a Hill fit model. A Schild plot
is the constructed and Kb is calculated.
29. Human erythrocytes and granulocytes were enriched from normal donor
peripheral blood by Isolymph (Gallard–Schlesinger Industries, Plainview, NY)
gradient centrifugation. The erythrocytes were removed using ACK lysing
buffer (Gibco, Carlsbad, CA). The mixed granulocyte population was pre-
incubated with vehicle (0.05% DMSO) or antagonists for 10 min at room
temperature prior to stimulation with PGD2 (600–0.003 nM at 1:3 dilution)
(Cayman Chemical Co, Ann Arbor, MI) for 10 min at 37 °C. The cells were fixed
using 1% final paraformaldehyde (Alpha Aesar, Ward Hill, MA) and were
analyzed on a FACS caliber (BD Biosciences, San Jose, CA) flow cytometer.
Leukocytes were gated on using forward/side scatter parameters. The FL2
positive cells (eosinophils) were then gated and their geometric mean of the
forward scatter was calculated. The geometric means were graphed using
GraphPad Prism 5 (GraphPad Software Inc, La Jolla, CA) and IC50 values were
calculated. The Kb values were calculated using the equation A/(R-1) where A is
the concentration of the inhibitor used. The value R = X/Y where X is the IC50
value of PGD2 in the presence of the inhibitor and Y is the IC50 value of PGD2
alone.
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