Fitc conjugated anti cd11b monoclonal antibody

Spleens were dissected from abdominal cavities of mice and were filtered through a 40-μm nylon strainer. A 0.83% aqueous solution of NH₄Cl was used to remove red cells. The resulting suspension of single splenic cells was stained with FITC-conjugated anti-CD11b monoclonal antibody (BD pharmingen, San Jose, CA), and the CD11b⁺ cells were collected by using a cell sorter (S3, Bio-Rad, Hercules, CA).

The phenotypes of the cells were classified by the expression of surface molecules. After the surface molecules were stained directly by a panel of fluorescence monoclonal antibodies, the cell surface molecular expressions of DC, myeloid-derived suppressor cells (MDSC) and regulatory T-cells (Treg) were analyzed by cytofluorography employing a Beckman Coulter NAVIOS flow cytometer (Beckman Coulter Co., Indianapolis, IN, USA). The fluorescence monoclonal antibodies included phycoerythrin (PE)-conjugated anti-CD40, CD80, CD86 and I-A^k (PharMingen, San Diego, CA, USA) for DC, phycoerythrin (PE)-conjugated anti-Gr-1 monoclonal antibodies and fitc-conjugated anti-CD11b monoclonal antibody (PharMingen, San Diego, CA, USA) for MDSC, and anti-foxp3 for regulatory T-cells (eBioscience, Thermo Fisher Scientific Inc., USA).

After the surface molecules were stained directly by PE-conjugated anti-Gr-1 monoclonal antibodies and FITC-conjugated anti-CD11b monoclonal antibody (1/100 × for rat monoclonal antibodies, and 1/50 × for mouse monoclonal antibodies; PharMingen, San Diego, CA, USA), the cell surface expression of markers on splenocytes was analyzed by flow cytometry employing a Coulter Epics Altra flow cytometer (Beckman Coulter Co., Miami, FL, USA). Isotype immunoglobulin was used as a control.