Acute astrocyte isolation

Acute isolation of astrocytes from adult C57BL/6J mice were performed according to previously described protocol [55]. First, an Adult Brain Dissociation Kit (cat. 130-107-677, Miltenyi Biotec) was used to dissociate cortical brain tissue into single-cell suspensions according to the manufacturer’s instructions. Adult mice were sacrificed under anesthesia with the brain quickly removed and stored in cold DPBS. Cortical tissue was dissected out (one brain per isolation) and kept in cold DPBS. Meninges were carefully removed, cortical tissue were minced and then transferred into gentleMACS C tubes (cat.130-093-237) which contained 1950 µL of enzyme mixture 1 solution and 30 µL of enzyme mixture 2 solution. The tightly closed C tubes were then incubated onto rotated gentleMACS Octo Dissociator with Heater for tissue dissociation (30 min). The obtained dissociated cells were re-suspended in DPBS, filtered with MACS SmartStrainer (70 µm) to remove any non-dissociated tissue and centrifuged for 10 min at 1000 rpm. The obtained pellets were re-suspended in cold DPBS and cell suspension was subjected to debris removal and red blood cell removal. The single cell-suspension obtained was then subjected to magnetic labeling with an Anti-ACSA-2 MicroBead Kit (cat. 130-097-678, Miltenyi Biotec) according to the manufacturer’s instructions. The cell pellets re-suspend were re-suspend in buffer and the cell suspension was blocked with 10–15 µL of FcR blocking reagent at 4 °C for 10 min. 10 µL of Anti-ACSA-2 MicroBeads per 10⁷ total cells was added in cell suspension, mixed well and incubated for 15 min at 4 °C in the dark. The cells were washed by adding 2 mL of AstroMACS separation buffer (cat. 130–117-336, Miltenyi Biotec) and centrifuged at 1500 rpm for 5 min to remove excess beads from the solution. The obtained pellets were re-suspended with buffer and loaded onto an MS Column (cat. 130-042-201, Miltenyi Biotec), which was placed in the magnetic field of a MACS magnetic cell separator. The column was washed with 3 mL buffer to remove unlabeled cells and the magnetically labeled ACSA-2-positive astrocytes cells were eluted as the positively selected cell fraction buffer after removing the column from the magnet separator. Then, RNA was isolated from purified astrocytes for Nanostring analysis.