Pe anti mouse acsa 2

Isolated brain cells were prepared from C57BL/6J mice. Tissue was digested by papain at 37 °C for 1 h (2 mg/mL, LS003119, Worthington, USA) in DMEM medium. Dispersed cells were filtrated with a nylon mesh (70 μm). The cells were resuspended in Percoll density gradient (30%, 17-0891-09, GE Healthcare, USA) and centrifuged (900×g) at 25 °C for 25 min. Next, the cells in the bottom were collected. After washing in PBS containing 2% FBS, the cells were blocked with FcR Blocking Reagent (130-092-575, Miltenyi Biotec). Astrocytes, microglial cells, neurons, and endothelial cells were marked by flow cytometry42 (link), 43 (link), 44 (link), 45 (link). Cells were stained with PE anti-mouse ACSA-2 (130-116-244, Miltenyi Biotec, Germany), FITC anti-mouse/human CD11b antibody (101205, BioLegend, USA), PerCp-cy5.5 anti-mouse CD45 antibody (561869, BD Pharmingen, USA), APC anti-mouse NCAM-1/CD56 allophycocyanin MAb (FAB7820A-100, R&D, USA), and Brilliant Violet 605™ anti-mouse CD31 (102427, BioLegend, USA). After staining, the samples were sorted by FACSAria II SORP (BD Biosciences, USA), and the data were analyzed using FlowJo_V10 (FlowJo). Samples were gated for ACSA-2⁺ (astrocytes), ACSA-2^–CD11b⁺CD45^dim (microglial cells), NCAM-1/CD56⁺ (neurons), and CD31⁺ (endothelial cells). The RNeasy®-Micro Kit (74004, QIAGEN, Germany) was used for RNA extraction.