Apc cy7 anti human cd14 clone m5e2

After PBMC isolation, cells were stained with the following antibodies to detect MDSCs: PE anti-human CD11b (clone ICRF44; BioLegend, San Diego, CA, USA), peridinin chlorophyll protein anti-human HLA-DR (clone L243; BioLegend), allophycocyanin (APC) anti-human CD33 (clone WM53; BioLegend), and APC-cy7 anti-human CD14 (clone M5E2; BioLegend). To detect chemokine receptors, the antibodies used were FITC anti-human CCR2 (clone K036C2; BioLegend), CXCR1 (clone 8F1/CXCR1; BioLegend), PE-cy7 anti-human CXCR2 (clone 5E8/CXCR2; BioLegend), and CXCR4 (clone 12G5; BioLegend). For MDSC detection, bone marrow cells were stained with FITC anti-mouse Ly-6G (clone 1A8; Miltenyi Biotec, Gladbach, Germany), APC anti-mouse Ly-6C (clone 1G7.G10; Miltenyi Biotec), and APC-cy7 anti-mouse/human CD11b (clone M1/70; BioLegend).
Staining was performed for 30 min at room temperature and cells were washed with FACS buffer. After washing, samples were collected on a BD FACSAria (Becton Dickinson, Franklin Lakes, NJ, USA). All statistical analyses were performed with FlowJo software (Tree Star Inc., Ashland, OR, USA).

PBMCs were isolated using density-gradient centrifugation over a Ficoll-Hypaque gradient (GE Healthcare, Piscataway, NJ, USA) to collect PBMCs. PBMCs were washed with PBS (137 mM NaCl, 2.7 M KCl, 10 mM Na₂HPO₄ and 2 mM KH₂PO₄, pH 7.4) and re-suspended in flow cytometry (FACS) buffer (0.1% BSA in PBS). The cells were stained with PE anti-human CD11b (clone ICRF44, Biolegend), peridinin chlorophyll protein (PerCP) anti-human HLA-DR (clone L243, Biolegend), APC anti-human CD33 (clone WM53, Biolegend) and APC-cy7 anti-human CD14 (clone M5E2, Biolegend) for 30 min to detect Mo-MDSCs. After washing, samples were isolated on BD FACSAria (Becton Dickinson) according to the manufacturer's protocol (Becton Dickson, Brea, CA, USA).