Anti acsa 2 microbead

The spinal cord, dissected from mice transcardially perfused with PBS, was dissociated at 37 °C for 15 min using the Neural Tissue Dissociation Kit—Postnatal Neurons (Miltenyi Biotec, Germany) by the gentleMACS Dissociator (Miltenyi Biotec, Germany). For isolation of microglia and astrocytes, myelin debris was removed by using Myelin Removal Beads II (Miltenyi Biotec, Germany). Purified cells were incubated with anti-CD16/CD32 antibodies (eBioscience, USA) for blocking Fc receptors, and then incubated with anti-CD11b microBeads (Miltenyi Biotec, Germany) for isolating microglia. CD11b-positive microglia were obtained by magnetic cell sorting through an LS column (Miltenyi Biotec, Germany). For isolating astrocytes, astrocyte-containing flow-through cells were incubated with anti-ACSA2 MicroBeads (Miltenyi Biotec, Germany), and then subjected to magnetic cell sorting through the LS column.

Astrocytes were isolated as previously described (52 (link)) with a few modifications. Briefly, mouse SC were minced into approximately 1 mm² pieces and digested using the Papain Dissociation System (Worthington Biochemical Corporation, catalog LK003153). Isolated cells were negatively selected in tandem first with Myelin Removal Beads II (Miltenyi Biotec, catalog 130-096-731) and second with anti-CD11b MicroBeads (Miltenyi Biotec, catalog 130-049-601) to collect the flowthrough fraction. To enrich astrocytes, the flowthrough cells were first treated with FcBlock (Miltenyi Biotec, catalog 130-092-575), then positively selected using anti-ACSA-2 MicroBeads (Miltenyi Biotec, catalog 130-097-678). Total RNA was prepared using TRI Reagent (MilliporeSigma, catalog 93289) and reverse-transcribed using qScript cDNA Mix (Quantabio, catalog 950048) to obtain cDNA. RT-qPCR assays were performed with SYBR FAST qPCR Master Mix (Kapa Biosystems, catalog KK4602), using primers indicated in Supplemental Table 2. Expression levels of target genes relative to an internal control, Actb, were calculated using the ΔΔCt method (53 (link)).

To perform cell type-specific proteomics and phosphoproteomics, astrocytes and neurons were separated from cortical single-cell suspensions as previously described^{62 (link)} with slight modifications. Single-cell suspensions were incubated with FcR Blocking Reagent (Miltenyi Biotec, 130-092-575) and Anti-ACSA-2 MicroBeads (Miltenyi Biotec, 130-097-678) for 10–15 min at 2–8 °C. Cells were spun down for 10 min at 300 × g at room temperature and resuspended in a minimal volume of PBS with 0.5% of BSA before passing through a LS Column. Flow-through was collected for further neuron separation, the LS Column that retained astrocytes was removed from the magnetic field and the ACSA-2 labeled astrocytes were eluted. Cells were pelleted from flow-through at 300 × g, 8 min, the supernatant carefully removed, and resuspended in a minimal volume of PBS with 0.5% of BSA. Suspensions were incubated with Non-Neuronal Cell Biotin-Ab cocktail (Miltenyi Biotec, 130-115-389) for 5 min and Anti-Biotin MicroBeads (Miltenyi Biotec, 130-090-485) for 10 min at 2–8 °C, respectively. Following incubation, suspensions were passed through a LS Column placed in a magnetic field and non-labeled neuronal cells were collected in flow-through.

Cerebella (either sex) derived from wild‐type and β‐actin‐GFP mice were dissected, pooled, dissociated, and blocked with FcR Blocking Reagent, mouse (Miltenyi Biotec). The Anti‐ACSA‐2 MicroBead Kit allowed for efficient enrichment of ACSA‐2⁺/GLAST⁺ cells to high purity using an MS column (both Miltenyi Biotec). The enrichment of ACSA‐2⁻/GLAST⁺ cells required a two‐step isolation protocol. First, single cells were labeled with Anti‐L1CAM‐PE (titer 1:2.5) (Cat# 130‐102‐865, RRID:AB_2655591) and then incubated with a mix of Anti‐PE MicroBeads (1:2.5) Cat# 130‐048‐801, RRID:AB_244373), Anti‐ACSA‐2 MicroBeads (1:2.5), Anti‐CD45 MicroBeads (1:10), Anti‐PSA‐NCAM MicroBeads (1:10), and Anti‐Ter119 MicroBeads (all MicroBead conjugates from Miltenyi Biotec) and processed using an LD column (Miltenyi Biotec) to deplete the unwanted cell types. The negative fraction was then incubated with Anti‐GLAST‐Biotin (Cat# 130‐095‐815, RRID:AB_10829314; Miltenyi Biotec) and then with Anti‐Biotin MicroBeads (Cat# 130‐091‐256, RRID:AB_244365; Miltenyi Biotec). Cells were subjected to magnetic cell separation to isolate GLAST positive cells.

The mice were deeply anesthetized with pentobarbital sodium and perfused transcardially with ice-cold saline. Whole brains without the olfactory bulb and cerebellum were immediately removed and washed with 4°C prechilled sterile phosphate-buffered saline. The brain tissue was mechanically and enzymatically dissociated using the Adult Brain Dissociation Kit (Miltenyi Biotec, #130-107-677) according to the manufacturer’s instructions. After myelin was removed using the cell debris removal buffers, the total cell pellet was resuspended in PBS containing 0.5% BSA, followed by the isolation of specific cell types. Briefly, astrocytes were isolated using anti-ACSA-2 MicroBeads (Miltenyi Biotec, #130-097-678) on a MACS^® MultiStand Separator, according to the manufacturer’s instruction. For RNA extraction, isolated cells were frozen using liquid nitrogen and stored at −80°C before use. Isolated cells from two mice were pooled for each sample.