Acsa 2 microbeads

Choosing the cerebral cortex of mice (1-3 days old) to extract primary astrocytes, the cortex was digested by 0.125% trypsin-EDTA (Gibco, Grand Island, NY, USA), then centrifuged and incubated in DMEM/F12 complete medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). After 9-11 days, the cells were shaken at 260 rpm at 37° C for 16 h to purify. Finally, purified astrocytes were obtained.
Choosing the cortex of fetal mice (16–18 days old) to isolate primary neurons, the culture flasks were pretreated with poly-d-lysine (PDL) (Sigma-Aldrich, St. Louis, MO, USA). The fragment was digested with 0.125% trypsin and grown with DMEM (Gibco, Grand Island, NY, USA) for 5 h, then replaced with neurobasal medium containing 2% B27 (Gibco, Grand Island, NY, USA) and 0.5 mmol/L glutamine (Sigma-Aldrich, St. Louis, MO, USA).
Choosing Adult Brain Dissociation Kit and ACSA-2 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for magnetic isolation of astrocytes from adult mice. The former was used to digest the cortex. Then, purified astrocytes were obtained. Astrocytes were incubated for 15 min at 4° C with ACSA-2 MicroBeads and separated from single-cell suspension in a magnetic field using MS columns, MACS MultiStand and QuadroMACS (Miltenyi Biotec, Bergisch Gladbach, Germany).

Microglia-enriched CD11b⁺ cells and ACSA-2⁺ astrocytes were isolated from one adult mouse model according to a previously described method with minor modifications^{87 (link)}. In brief, utilizing the same method used to create the primary microglia culture, CD11b⁺ cells were isolated by anti-CD11b MicroBeads (cat # 130-093-634, MACS Miltenyi Biotec, Auburn, CA). CD11b⁺ cells-removed cell pellets were resuspended and incubated with FcR blocking reagent for 10 min, followed by ACSA-2 MicroBeads (cat # 130-097-678, MACS Miltenyi Biotec, Auburn, CA) for 15 min, and then loaded onto LS columns where they were separated on a quadroMACS magnet. ACSA-2⁺ cells were flushed out from the LS columns, and ACSA-2⁺ cells-removed cell pellets were used as remaining cells.