Xbd173

Primary microglia at the bottom and 661W cells on top were separated in a trans-well culture with 0.4 μm inserts. As a basal culture medium, DMEM supplemented with 10% FCS, 1% Pen/Strep, was used. Primary microglia were seeded at a density of 2.5 × 10⁵ cells/well in a 24-well plate and 661W cells at a density of 2.5 × 10⁴ cells/well in 0.4 µm trans-well inserts. After 4 h, trans-wells with 661W cells were removed, placed in a new sterile 24-well plate and primary microglia were stimulated with 661W photoreceptor cell debris by synchronization at 450 × g for 5 min at 4 °C. Where indicated, primary microglia were treated with 1 mM l-ascorbic acid (Sigma-Aldrich), 50 µM XBD173 or 50 mM NAC (Sigma-Aldrich). After incubation for 15 min at 37 °C, trans-wells with 661W cells were transferred back to the primary microglia and the trans-well co-culture was incubated for another 24 h.

PK11195 (Tocris), Ro5-4864 (Sigma-Aldrich), and XBD-173 (Sigma-Aldrich) were all prepared in 1% DMSO, 99% saline solution. Intraperitoneal injections (i.p.) were given at a volume of 0.1 ml per 10 g of body weight. The doses of 1 and 5 mg/kg were selected to be lower than prior studies with the tested ligands examining behavioral effects, attempting to fall below the established doses for sedation, and well below the dose needed for proconvulsant activity by Ro5-4864 (30 + mg/kg). Since the ligands do not all share a binding site (Jaremko et al., 2014 (link), Jaremko et al., 2015 (link)), we did not attempt to base dosing on K_i, but rather focused on the behavioral impacts of the ligands.

WT MA-10 and STAR KO cells (1×10⁴ per well) were plated on 96-well plates in triplicate for 24 h. Before stimulation, medium was removed, each well was washed with phosphate-buffered saline, and serum-free medium was added with one of the following: 50 ng/mL human chorionic gonadotropin (hCG; National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA, USA), 1 mM dibutyryl cyclic AMP (dbcAMP; Sigma), 50 μM 22(R)-hydroxycholesterol (Sigma, St. Louis, MO, USA), 50 μM XBD173 (Sigma), 50 μM FGIN-1-27 (Cayman Chemical, Ann Arbor, MI, USA), 1-oleoyl-2-acetyl-sn-glycerol (Cayman Chemical, Ann Arbor, MI, USA), U73122 (Cayman Chemical, Ann Arbor, MI, USA), or 1 mM calphostin C (Cayman Chemical, Ann Arbor, MI, USA). After 2 h incubation at 37 °C, the medium was collected to measure steroid production. The remaining cells were lysed with 0.1 N sodium hydroxide for protein measurements. Steroid production was measured using the Progesterone ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA). Steroid production data were normalized to protein contents.