Pe conjugated anti mouse cd11c

Macrophages grown in 6-well tissue culture plates were infected with FMDV as described earlier and cells were harvested at 0, 4, 12 and 18 hpi. After treatment, cells were washed with PBS and detached from culture plates by adding PBS containing EDTA (5 mmol/L) and incubated at 4 °C for 10 min, before thorough pipetting. 1 × 10⁶ cells per sample were used for analysis by Cytomics FC500 flow cytometer (Beckman Coulter, USA) and stained with the following antibodies: APC-conjugated anti-mouse F4/80 antibody, PE-conjugated anti-mouse CD11c and FITC-conjugated anti-mouse CD206 (all antibodies were from BioLegend, USA). Antibodies were diluted in FACS buffer (PBS containing 5 % FBS, pH 7.4). Each antibody was incubated at 4 °C for 15 min in the dark. Cells were washed twice with FACS buffer between each antibody incubation. After washing, cells were resuspended in FACS buffer and run on a Cytomics FC500 flow cytometer (Beckman Coulter, USA). M1 macrophages were identified as F4/80-positive/CD11c-positive while M2 macrophages were identified as F4/80-positive/CD206-positive. The data was analyzed by CXP Software Version 2.2 (Beckman Coulter, USA).