Dnaase 1

Primary cultures of astrocytes were prepared from P1–P4 mouse brains. Cerebral cortices were dissected and dissociated in 15 mL tubes at 2 mg/mL papain (Worthington) in sterile Hybernate-A (Gibco) under agitation at 37 °C for 15 min. The papain was then inactivated with a mixture of ovomucoid solution and DNAase I (both Worthington). The cell suspension was then washed twice using pre-warmed Neurobasal media and centrifugation at 17×g for 5 min. The cell suspension was then strained through a 100 μm cell strainer (CellTreat), resuspended in Neurobasal medium supplemented with 10% (v/v) FBS and 10 U/mL penicillin and streptomycin (Gibco). Cells were then plated onto T-75 flasks (Falcon) pre-coated with poly-D-lysine at 5 μg/mL (Sigma) at around 10,000 cells per cm². Cells were maintained in T-75 flasks at 37 °C in a humidified 5% CO₂ chamber for 2 weeks before frozen down as stock for future cell culture experiments.

Clonogenic assays were performed on murine tumoural pituitaries as described (Gaston-Massuet et al. 2011 (link), Carreno et al. 2017 (link), Haston et al. 2017 (link)). Pituitaries were dissected using aseptic forceps and the posterior pituitary was removed. After mincing with forceps, the remaining tissue was placed into 200 µL of enzyme mix, which consisted of Hanks’ Balanced Salt Solution (HBSS, Gibco), 0.5% w/v Collagenase (Worthington), 50 µg/mL DNAase I (Worthington), 1% Fungizone and 0.1× trypsin (Sigma), for 4 h in a 37°C water bath. HBSS was added to make a final volume of 500 µL post incubation and the solution was triturated into a single-cell suspension. Once single-cell suspensions were achieved, 9.5 mL of HBSS was added and the cells spun down for 5 min at 200 g. Cells were re-suspended in growth medium, which consisted of DMEM/F12, 5% FCS, 1% PenStrep, 20 ng/mL human recombinant bFGF (R&D Systems) and 50 ng/mL cholera toxin. Cells were plated at clonal density in a six-well plate at 2000, 4000 and 8000 cells per well. Fresh bFGF was added after 2 days, and medium was then changed on the third day and every 3 days after colony establishment. Colony counting was conducted after 7 days in culture. Colonies were washed with PBS and fixed for 20 min with 4% PFA, washed again in PBS and stained with Harris haematoxylin for 15 min at room temperature.