Anti ly6c pe cy5

Leukocytes were suspended in cold PBS (1 × 10⁷ cells/ml), incubated with anti-mouse CD 16/32 (eBioscience, San Diego, CA, 1 μl/ml), then flurochrome-conjugated antibodies were added (0.5 μg/10⁶ cells/0.1 ml) at 4°C for 30 minutes. Flow antibodies used for these studies included anti-F4/80-FITC (clone BM8, eBioscience), anti-Ly6G-PE (clone 1A8, BD Biosciences), anti-Ly6C-PE Cy5.5 and appropriate isotype controls (eBioscience and BD Biosciences). Neutrophils are identified as Ly6G⁺F4/80⁻ cells, macrophages are identified as Ly6C⁻F4/80⁺ cells, and monocytes are identified as Ly6C⁺F4/80⁺ cells. Samples were run on an Accuri C6 flow cytometer (BD Biosciences, San Diego, CA). Data were analyzed using Accuri C6 software.

Leukocytes obtained by peritoneal lavage were suspended in phosphate buffered saline (PBS) (1 × 10⁷ cells/ml) and incubated with anti-mouse CD16/32 (eBioscience, 1 μl/ml) for 5 minutes to block nonspecific Fc receptor–mediated antibody binding. One million cells were then transferred into polystyrene tubes, incubated with fluorochrome-conjugated antibodies or isotype controls (0.5 μg /tube), washed with 2 ml of cold PBS, centrifuged at 300xg for 10 minutes and resuspended in 250 μl cold PBS. Samples were run immediately on an Accuri C6 flow cytometer (BD Biosciences, San Diego, CA)(8 (link)). Data were analyzed using Accuri C6 software.
Antibodies included anti-Ly6G-PE, anti-Ly6C-PE Cy5.5, anti-F4/80-FITC, and appropriate isotype controls (EBioscience, San Diego, CA). Neutrophils were identified as F4/80⁻Ly6G⁺, macrophages as F4/80⁺Ly6C⁻, and monocytes as F4/80⁺Ly6C⁺.