Sc 51322

Before all experiments, astrocyte medium was exchanged for low serum media ±1 μg/mL LPS. For exogenous PGE₂ treatment, human PGE₂ (Cayman Chemical, Ann Arbor, MI) at 1, 2, 4, 8, 16 or 20 ng/mL was added immediately, or 6 h after LPS. For agonist studies, iloprost (EP1, Cayman Chemical, Ann Arbor, MI), butaprost (EP2, Cayman Chemical, Ann Arbor, MI), sulprostone (EP3, Cayman Chemical, Ann Arbor, MI), or CAY10598 (EP4, Cayman Chemical, Ann Arbor, MI) was added at 10 nM, 100 nM, 1 μM, or 10 μM. For antagonist studies, 20 ng/mL PGE₂ was added along with SC-51322 (EP1, Cayman Chemical, Ann Arbor, MI), PF-04418948 (EP2, Cayman Chemical, Ann Arbor, MI), L-798,106 (EP3, Sigma–Aldrich, St. Louis, MO), or L161,982 (EP4, Cayman Chemical, Ann Arbor, MI) at 10 nM, 100 nM, 1 μM, or 10 μM. For antagonist blocking studies, monolayer or encapsulated MSCs were co-cultured with astrocytes and antagonists were added concurrently at doses determined by antagonist studies (10 μM SC-51322, 10 μM PF-04418948, 10 μM L-798,106, or 1 μM L-161,982). All cultures were returned to incubators at 37°C in 5% CO₂, and media supernatants were collected 24 h post-LPS stimulation.

DCs were positively selected from the spleen using an autoMACS separator and CD11c magnetic beads (Miltenyi Biotech). The purity of these was confirmed to be >95% by flow cytometry. Splenic CD11c⁺ cells were placed in 24-well plates at a density of 5×10⁵ per well and cultured in 500 μL RPMI1640 medium supplemented with 10% fetal bovine serum for 24 hours. PGE₂, EP1 receptor antagonist SC-51322, EP3 receptor antagonist DG-041, EP1 or EP3 receptor agonists 17-phenyl-trinor-PGE2 (Cayman Chemical, Ann Arbor, MI), sulprostone (Cayman Chemical, Ann Arbor, MI), MB-28767 (a gift from Dr. M.P.L. Caton Rhone-Poulenc), and ONO-AE-248 were applied as indicated below. DG-041 and ONO-AE-248 were provided by the Vanderbilt Institute of Chemical Biology Synthesis Core.