Anti acsa 2 microbead kit

Astrocytes were isolated as described previously [17 (link), 18 (link)]. Mice were sacrificed by decapitation, and the brains were immediately removed to isolate CTX and CC tissue. Brains were diced into 1 mm³ pieces using a scalpel blade. The tissues were incubated in an oxygenated papain solution containing 100 units of papain (Worthington Biochemical, LS003126), 2 mg of l-cysteine (Sigma-Aldrich, C7880) and DNase I (Sigma-Aldrich, D4527) at 34 °C for 40 min. Following tissue digestion, papain was inactivated using a low-ovo solution containing BSA, PBS, Trypsin inhibitor (Worthington Biochemical, LS003086). The brain tissues were dissociated by gently pipetting in low-ovo solution. Following centrifugation, the cell pellet was suspended in PBS and counted. Then, the cell suspension was subjected to magnetic labeling with an Anti-ACSA-2 MicroBead Kit (Miltenyi Biotec, #130–097-679) according to the manufacturer's instructions. Briefly, the cell suspension was blocked with 5–10 µl of FcR blocking reagent per 10⁷ total cells at 4 °C for 10 min; 5–10 µl of Anti-ACSA-2 MicroBeads per 10⁷ total cells was added and incubated at 4 °C for 15 min. The cells were washed by adding 1–2 ml of PBS buffer per 10⁷ total cells and resuspended in 500 µl PBS buffer per 10⁷ total cells. The cell suspension was applied to an MS column (Miltenyi Biotec, #130-042-201) to get purified astrocytes.

Mouse adult brain tissue was dissociated with an adult mouse brain dissociation kit (Miltenyi Biotec, #130‐107‐677). Dissociated cells, after removal of debris and red blood cells, were separated magnetically with an astrocyte‐specific anti‐ACSA‐2 Microbead Kit (Miltenyi Biotec, #130‐097‐678). We confirmed the identity of the isolated fractions by flow cytometry against astrocytic‐specific marker ACSA‐2 (Miltenyi Biotec, #130‐117‐535). The flow cytometry was performed in BD FACS CANTO II flow cytometer (BD Biosciences). The software used for data acquisition was BD FACSDIVA and the software used for data analysis was FlowJo_v10.7.2.

Peripherally mediated neuroinflammation was induced as previously described (27 (link), 28 (link)). In brief, 5 mg/kg LPS from E. coli O111:B4 (InvivoGen, tlrl-eblps) were dissolved in 200 μL of 1× PBS and injected i.p. into 8- to 12-week-old female C57Bl/6J mice. Sterile PBS served as the vehicle control. Mice were sacrificed after 24 hours, and ACSA2⁺ astrocytes were isolated from the cortex of LPS- and PBS-injected mice using the anti–ACSA-2 MicroBead Kit (Miltenyi, 130-097-678) after single-cell preparation, as described below.

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.