Inos apc

To evaluate immune cells in mice brain, brain tissues were cut into pieces and digested with 150 μL collagenase (1 mg/mL) and 250 μL DNase (10 mg/mL) for 1 h at 37 °C. Next, cells were harvested and passed through 70 μm strainer. Red Blood Cell Lysis Solution (Beyotime, China) was used to deplete erythrocytes. To evaluate adherent cells in culture dish, cells were digested by 0.05% trypsin for 3 min and separated as single cell suspension. For surface staining, 1 × 10⁶ cells were blocked with anti-FC receptor antibody at 4 °C for 15 min, then stained with indicated antibodies at 4 °C for 30 min. For intracellular staining, cells were fixed and permeabilized using the intracellular staining kit (ThermoFisher, USA). Flow cytometry was performed on a CytoFLEX (Beckman Coulter, USA) and analyzed using FlowJo software (Treestar, USA). The antibodies used in flow cytometry were: CD86 PE (#12-0861-83), CD206 FITC (#MA5-16870), CD11b APC (#RM2805), Iba1 (#PA5-27436), TNF-α PE-CY7 (#25-7321-82), iNOS APC (#17-5920-82), IL-10 PE (#12-7101-82), Arg1 PE-CY7 (#25-3697-82), CD4 PE (#12-0041-82), CD3 APC (#17-0038-42), CD8 PE (#MA5-17849), CD19 PE (#12-0199-42), CD20 FITC (#11-0209-42), CD56 PE (#12-0567-42), CD25 PE-CY7 (#25-0251-82) and FoxP3 PerCP-CY5.5 (#45-5773-82) were all from ThermoFisher (USA).

Samples were incubated with an Fc receptor blocker (CD16/32, BD Bioscience) to reduce nonspecific antibody binding. Freshly isolated samples were resuspended in staining buffer (R&D Systems) and stained with F4/80‐FITC (eBioscience) for 30 min at 4 °C. For intracellular staining, we used the Intracellular Fixation and Permeabilization Kit (BD Bioscience); this was used in accordance with the manufacturer’s instructions. Cells were then washed and stained with iNOS-APC (eBioscience) and CD206-PE (eBioscience). Flow cytometry was performed using a FACS Aria flow cytometer (BD Bioscience) and data were analyzed with FlowJo V10.6.2 software (TreeStar, Ashland, OR).