Isolation of Primary Astrocytes

Astrocytes were acutely isolated as previously described (Holt and Olsen, 2016 (link); Kahanovitch et al., 2018 (link); Stoica et al., 2017 (link)). Following dissociation, microglia and myelin were first removed from the cell suspension. Cells were incubated for 15 min at 4°C with 15 µL of Miltenyi Biotec’s Myelin Removal Kit and Cd11b⁺ MicroBeads. The suspension was then applied to a prepped LS column, washed three times, and the flow-through collected. This flow through was subsequently used to isolate astrocytes utilizing Miltenyi Biotec’s ACSA-2⁺ MicroBead kit. The cell suspension (in 150 µL 0.5% fatty-acid free BSA in PBS) was incubated at 4°C for 15 min with 15–20 µL FcR blocker, followed by a 15 min incubation with 15–20 µL ACSA-2 microbeads. Cells were applied to a prepped LS column. Astrocytes were eluted from the LS column after three washes, with 5 mL buffer and the supplied plunger.