Pe conjugated anti cd11b mab

To examine MDSCs in the spleen and tumor sites, suspended cells were stained with an allophycocyanin (APC)‐conjugated anti‐CD45 mAb (BioLegend), phycoerythrin (PE)‐conjugated anti‐CD11b mAb (BioLegend), fluorescein isothiocyanate (FITC)‐conjugated anti‐Gr‐1 mAb (R&D Systems), and PE/Cy7‐conjugated anti‐Ly6C mAb (BioLegend). To examine T cells in tumor tissues, suspensions of cells from tumor tissues were stained with an APC‐conjugated anti‐CD45 mAb, PE‐conjugated anti‐CD4 mAb (BioLegend), and FITC‐conjugated anti‐CD8 mAb (Southern Biotech). To assess the proportions of tumor peptide‐specific CD8⁺ T cells in draining lymph nodes (LNs), axially draining and inguinal LN cells were stained with a PE‐conjugated tetramer of an H‐2L^d‐binding AH1 peptide and FITC‐conjugated anti‐CD8 mAb. Stained cells were analyzed using a FACSCalibur flow cytometer (Becton‐Dickinson).

To analyze tumor‐infiltrating immune cells, tumor tissues were resected from mice individually, 22 days after tumor inoculation (4 days after the second chemotherapy treatment). The tumors were minced, placed on glass slides, and passed through gauze mesh and nylon mesh immediately before flow cytometry. The following mAbs were used: APC‐conjugated anti‐CD45 mAb (BioLegend), FITC‐conjugated anti‐Gr‐1 mAb (BioLegend), PE‐conjugated anti‐CD11b mAb (BioLegend), FITC‐conjugated anti‐CD8 mAb (BioLegend), PE‐conjugated anti‐CD4 mAb (BioLegend), and PE/cy7‐conjugated anti‐Ly6C mAb (BioLegend). Similarly, spleen cells of tumor‐bearing and treated mice were analyzed. Analysis was performed using FACSCalibur (Becton‐Dickinson).