Anti cd45 bv421

After 24 hours post injection, mice were killed by CO₂ asphyxiation. To evaluate levels of central nervous system (CNS)-associated phagocytes, hippocampal slices were isolated from mouse brains, suspended in DMEM supplemented with 10% FBS, and dissociated to single cells using a Pasteur pipette. Cells were then filtrated using a cell strainer, following which they were resuspended in PBS containing 10% FBS and stained with anti-CD45-BV421 (1:50, BD Biosciences) and anti-CD11b-PE antibodies (1:50, eBioscience). Cells were then analyzed via flow cytometry, and CD45⁺CD11b⁺ cells were considered as CNS-associated phagocytes (Cazareth et al., 2014 (link)). To quantify the levels of proinflammatory cytokines in the brain, hippocampal slices or whole brain tissues were homogenized using 25 mM Tris-Cl (pH 7.5) containing 1% NP-40, 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitors. The samples were then sonicated and centrifuged. The soluble fractions were normalized based on protein concentration using Bradford assay and then used to quantify the levels of proinflammatory cytokines via ELISA.

Tumor-infiltrating immune cells and splenocytes were analyzed using flow cytometry. Anti-CD45-BV421 (BD bioscience), anti-CD8-FITC (BioLegend), and anti-CD4-FITC (BD Bioscience) antibodies were used to characterize the cells (1×10⁶) using the FACSVerseTM system (BD Bioscience, Piscataway, NJ, USA) in accordance with the manufacturer’s protocol.