Myelin removal kit

Astrocyte isolation was performed according to Holt and Olsen (2016) (link). Briefly, mice were anesthetized with CO₂ and rapidly decapitated, and their cortices were microdissected in ice-cold cutting solution (120 mm NaCl, 3 mm KCl, 2 mm MgCl₂, 0.2 mm CaCl₂, 26.2 mm NaHCO₃, 11.1 mm glucose, and 5 mm Hepes), supplemented with AP5 (3 mm) and CNQX (3 mm) and bubbled with 95% oxygen. The tissue was minced and enzymatically dissociated with a Papain Dissociation kit (Worthington) following manufacturer’s instructions. After dissociation, myelin debris and microglia were removed using a Myelin Removal Kit (Miltenyi Biotec) and Cd11b⁺ Microbeads (Miltenyi Biotec), respectively. Astrocytes were then acutely isolated using anti–ACSA-2⁺ (astrocyte cell surface antigen 2) MicroBead kit (Miltenyi Biotec). The ASCA-2⁺ epitope is from the astrocyte-specific β2 subunit of the sodium potassium exchanger (Batiuk et al., 2017 (link)). The anti–ACSA-2⁺ antibody is highly specific for astrocytes (Holt and Olsen, 2016 (link)), is robustly expressed in astrocytes (Kantzer et al., 2017 (link)), and is used as a first choice for isolating astrocytes (Batiuk et al., 2017 (link)). Manufacturer instructions were generally followed, except incubation times were extended to 25 min and total volume of microbeads was increased to 20–40 μl.

The cerebellum was macrodissected for astrocyte isolation using magnetic-activated cell sorting (MACS) [16 (link)]. Briefly, the cerebellum was mechanically dissociated using a scalpel, followed by enzymatic dissociation using the Neural Tissue Dissociation kit (P) (Miltenyi Biotec, Cologne, Germany), according to the manufacturer’s instructions. Myelin and cell debris were removed using the Myelin removal kit (Miltenyi Biotec), followed by microglia removal using Cd11b microbeads (Miltenyi Biotec). Finally, astrocytes were isolated using anti-astrocyte cell surface antigen (ACSA)-2 beads (Miltenyi Biotec). All the separation steps were performed using the AutoMACS Pro Separator equipment (Miltenyi Biotec).

Meninges discarded tissue samples were transported immediately to the laboratory for processing. The tissue was dissociated (Brain Dissociation kit, Miltenyi). The dissociated cells underwent myelin removal (Myelin removal kit, Miltenyi) and CD11b+ cells were further purified (CD11b microglia Microbeads, Miltenyi). All kits were used according to the manufacturers instructions. The cells were then surface stained with fluorescently conjugated antibodies for microglial markers: CD11b-APC, CD45-Bv605 and P2RY12-Bv421, CD26-FITC, and CD36-PE (Biolegend). The cells were incubated with antibodies for 30 min on ice, washed, resuspended in 500 μL staining buffer, and acquired on a FACS Fortessa (BD Biosciences). Results were analyzed using FlowJo software.