All antibodies were purchased from BD Biosciences (USA) except for phycoerythrin (PE)-conjugated anti-CD33 (Pharmingen, USA), PE-conjugated TCR-ζ mAb (Beckman Coulter Immunotech SAS, France) and fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (Sungene Biotech, China).
The peripheral blood cells were analyzed using protocols previously described by our team, with minor modifications (Zou et al., 2009 (link)). Anti-lin-1-FITC, anti-HLA-DR-peridin chlorophyll protein, anti-CD11b-allophycocyanin (APC), and anti-CD33-PE were used to characterize MDSCs. For phenotypic staining ex vivo, fresh heparinized peripheral blood (200 μl) was labeled with the above-mentioned cocktail of antibodies; matched isotype control antibodies were used as negative controls. After incubation for 30 min at 4°C in the dark, FACS™ lysing solution (BD PharMingen) was added to lyse the red blood cells. After washing twice with phosphate-buffered saline (PBS), the cells were fixed in 1% paraformaldehyde, and four-color flow cytometric analysis was performed using a FACSCalibur flow cytometer (BD Biosciences, USA) and Flowjo software (TreeStar, USA) (Zhang et al., 2010 (link)). At least 200,000 events were acquired per run.
The peripheral blood cells were analyzed using protocols previously described by our team, with minor modifications (Zou et al., 2009 (link)). Anti-lin-1-FITC, anti-HLA-DR-peridin chlorophyll protein, anti-CD11b-allophycocyanin (APC), and anti-CD33-PE were used to characterize MDSCs. For phenotypic staining ex vivo, fresh heparinized peripheral blood (200 μl) was labeled with the above-mentioned cocktail of antibodies; matched isotype control antibodies were used as negative controls. After incubation for 30 min at 4°C in the dark, FACS™ lysing solution (BD PharMingen) was added to lyse the red blood cells. After washing twice with phosphate-buffered saline (PBS), the cells were fixed in 1% paraformaldehyde, and four-color flow cytometric analysis was performed using a FACSCalibur flow cytometer (BD Biosciences, USA) and Flowjo software (TreeStar, USA) (Zhang et al., 2010 (link)). At least 200,000 events were acquired per run.