Rat anti mouse cd16 32 2.4g2

Single-cell suspensions isolated from either, brain, BS, spleen, or cervical lymph nodes (CLN) were blocked with a 10% mixture of normal mouse, rat and horse serum, and rat anti-mouse CD16/32 (2.4G2, BD PharMingen) for 15 min prior to incubation with antibodies (Abs) to determine cell surface expression of various markers. Phycoerythrin (PE), FITC, and allophycocyanin-conjugated Abs specific for F480 (BM8), CD11b (M1/70), CD8 (53-6.7), and CD4 (RM4-4) were obtained from eBioscience (San Diego, CA). FITC, PE, and PerCP-conjugated Ly6-G (1A8), Ly6-C (AL-21), and CD45 (30-F11) were obtained from BD PharMingen (San Jose, CA). All isotype controls were obtained from eBioscience. To determine cell surface expression, antibody-labeled cells were acquired on a BD Fortessa Analyzer (BD Biosciences, San Jose, CA) and flow cytometry analysis was performed using FlowJo software (Tree Star Inc.). Doublets were excluded from live cell populations. CD45 was used to distinguish bone marrow-derived CD45^high leukocytes, from CD45^int CD11b⁺ microglia and CD45^neg neural/glial cells. Leukocyte subsets, calculated as a fraction of the live CD45^high-infiltrating population in the BS, were expressed as percentages.

BM-derived mφs (BMDMs) were trypsinized and resuspended in flow cytometry buffer (PBS with 2% FBS). Following this, 1 × 10⁶ cells were blocked with 0.5 μg rat anti-mouse CD16/32 (2.4G2; BD Pharmingen) in a volume of 100 μl for 30 min on ice. Cells were then stained with the following Abs: 5 μg/ml eFluor450 anti-F4/80 (BM8; eBioscience), 1.25 μg/ml PE Cy7 anti-CD115 (25-1152; eBioscience), or 1.2 μg/ml eFluor450 anti-CD11b (M1/70; eBioscience). Flow cytometry was performed with a Fortessa LSR II (BD Biosciences) and analyzed using FlowJo software.