Pituitary Dissociation and Immune Depletion

Whole pituitaries were dissected from wild-type C57BL/6 female mice at 9–10 weeks of age. Whole pituitaries were isolated into ice-cold PBS and then dispersed by incubation with 0.25% collagenase Type IV and 0.25% trypsin–EDTA (1x) (Life Technologies) as previously described (23 (link)). For immune depletion studies, dispersed pituitary was divided in half, with one half being subjected to depletion of total CD45+ immune cells using Miltenyi Biotech anti-C45 micro beads according to protocol. The cells (dispersed pituitary or immune cell depleted pituitary) were plated on poly-l-lysine (Sigma-Aldrich Inc.) coated Nunc 96-well plates (Thermo Fisher Scientific) at a density of 1.5 × 10⁶ cells per cm². The cells were cultured for 24 h at 37°C and 5% CO2 in high-glucose HEPES-buffered DMEM with 10% FBS prior to experimentation. After pituitary cultures equilibrated they were treated serum starved for 16 hrs, followed by a change in media and 30 min treatment with or without GnRH.