Anti asca 2 microbeads

Microglia, neurons, and astrocytes were isolated from mouse hemibrains by MACS, adapted from previously described methods (Bordt et al., 2020 ). For comparison of Grn in neurons, microglia, and astrocytes, a total of 6 mice were used. Brains from these mice were sliced at the midline to generate a total of 12 hemibrains, with each hemibrain assigned to isolation of one cell type. Mice were transcardially perfused with 0.9% saline, brains were removed, sliced into hemibrains, and immediately processed for MACS. Tissue was dissociated by digestion with 2 mg/mL Collagenase A (MilliporeSigma) and 0.5 mg/mL DNase I (MilliporeSigma) and trituration with a series of fire-polished pipettes (Bordt et al., 2020 ). After dissociation, samples were treated with debris removal solution (Miltenyi #130–109–398) according to the manufacturer’s instructions. Microglia were isolated using anti-Cd11b microbeads (Miltenyi #130–049–601), astrocytes were isolated using anti-ASCA-2 microbeads (Miltenyi #130–097–678), and neurons were isolated by negative selection using a mouse neuron isolation kit (Miltenyi #130–115–390). Microbead-bound cells were separated using a Quad-roMACS separator and LS columns (Miltenyi #130–091–051). After separation, cells were pelleted by centrifugation, then processed with Trizol reagent (ThermoFisher) for RNA isolation.

The whole brain of mice embryos (E16) was harvested and dissociated with Neural Tissue Dissociation Kit (Miltenyi Biotech Inc., Auburn, CA) following the manufacturer’s instructions. Astrocytes were purified with anti-ASCA-2⁺ microbeads (Miltenyi Biotech Inc., Auburn, CA) following the manufacturer’s MACS instructions (30 (link)). The purified astrocytes were centrifuged at 300 g for 10 min, and then resuspended with D-MEM/10% FBS for cell culture. Microglia cells were purified with anti-CD11b microbeads (Miltenyi Biotech Inc., Auburn, CA) following the manufacturer’s MACS instructions. The purified microglia cells were centrifuged at 300 g for 10 min, then resuspended with D-MEM/10% FBS plus 5 ng/ml M-CSF (PeproTech), and seeded on 60-mm dishes at a density of 1×10⁶/dish. After 7 days, cultures were trypsinized and replated in Petri dishes. Cells from cultures that had been passaged once were used as microglia cells.