Isolation and Culture of Neonatal and Adult Astrocytes

Neonatal astrocytes were prepared from brains of newborn C57BL6 wild type mice according to published protocols [25 (link), 26 (link)]. Briefly, primary mixed glial cultures were established from the forebrains of 1 day-old male and/or female pups, mechanically dissociated and digested with 0.25% trypsin solution. Mixed cultures were maintained in complete astrocyte medium (DMEM-high glucose medium containing 1% antibiotics (Pen/Strep solution), 200 mM l-Glutamine, 100 mM Sodium Pyruvate and 10% FCS (all from Euroclone, Italy)). Ten day-old primary cultures were vigorously shaken to discard microglia and oligodendrocytes. The remaining adherent cells were detached and, following an adhesion step, non adherent cells were reseeded on poly-d-lysine (Sigma) coated flasks. Adult astrocyte cultures were established from 2 months-old male and/or female C57BL6 wild type mice. Brains were extracted and dissociated by combining enzymatic and mechanical dissociation using Adult Brain Dissociation Kit and the gentleMACS™ Dissociator (both from Miltenyi Biotec). After dissociation, myelin, cell debris and erythrocytes were removed and remaining cells were seeded on poly-d-lysine coated flasks in complete medium. Purity of astrocyte cultures was > 95% according to GFAP immunofluorescence.