Astromacs medium

Brain astrocytes were isolated at PND 150 from naïve Oprm1 icKO and control mice (for Western blotting and the metabolic flux assay) and from mice that underwent the CPA test (for metabolic flux assay). Whole brains were dissected and placed in Dulbecco’s phosphate-buffered saline containing Ca²⁺, Mg²⁺, D-glucose, and pyruvate (14287080, Thermo Fisher Scientific, Waltham, MA, USA). Astrocytes were isolated according to the “Isolation and cultivation of astrocytes from adult mouse brain” Miltenyi Biotec protocol for the Adult Brain Dissociation kit (130-107-677, Miltenyi Biotec, Bergisch Gladbach, Germany) and anti-ACSA-2 MicroBead kit (130-097-678, Miltenyi Biotec). After magnetic sorting, cells were centrifuged for 5 min at 300× g at 4 °C. Pellets were dissolved in cell lysis buffer (9803, Cell Signaling Technology, Danvers, MA, USA) for Western blotting or in AstroMACS medium (130-117-031, Miltenyi Biotec) for subsequent cultivation.

For isolation of primary cortical astrocytes from adult mice, cortices from two 8–12 week old GFAP-CreERT2;p53^flox/flox;LSL-tdTomato mice were pooled and dissociated using the Adult Brain Dissociation Kit (Miltenyi 130-107-677), according to manufacturer’s instructions. Astrocytes were purified from cell suspension after removal of debris and red blood cells with ACSA-2 MicroBeads (Miltenyi 130-097-678) according to manufacturer’s instructions and plated onto two wells of a PLL and laminin coated 12 well plate in AstroMACS Medium (# 130-117-031). The following day, plates were gently washed with media to remove debris. Media was replaced every 3 days thereafter. While in culture, adult astrocytes showed very limited divisions, as previously described.⁹² (link)

Cell culture plates (24-well or 96-well as indicated; BD Biosciences) were prepared two days prior to astrocyte isolation. First, wells were coated with 20 µg/ml poly-l-lysine (PLL) (Sigma, 2636-25MG) in PBS overnight at 37 °C, 5% CO₂. The next day, wells were washed twice with PBS and subsequently coated with 2 µg/ml laminin in PBS (Sigma, L2020) overnight at 37 °C, 5% CO₂. For immunofluorescence, cells were plated onto 13 mm coverslips (VWR) coated with 0.5 mg/ml PLL and 10 µg/ml laminin. For calcium imaging, cells were plated onto glass bottom dishes (MatTek, P35G-1.5-14-C) coated with 0.5 mg/ml PLL and 10 µg/ml laminin. Astrocyte isolation was performed as described above under sterile conditions. ACSA-2 labeled cells were flushed from the MS column with pre-warmed AstroMACS medium (Miltenyi Biotec, 130-117-031) supplemented 50 U/ml penicillin/streptomycin (Sigma, P0781-20ML) and 0.25% l-glutamine (0.5 mM; Thermo Fisher, 25030-024). Neonatal and adult astrocytes were cultured using the same medium composition. Cells were plated at 100,000 cells/24-well or glass dish or 25,000 cells/96-well onto the middle of the well in a droplet of AstroMACS medium and incubated at 37 °C, 5% CO₂ for 1–3 h before filling up the medium. The medium was changed every three days and grown for 7–10 days before use.