Anti lineage cocktail 1 lin1 fitc

Blood samples for the analyses of immune cell populations were obtained at the time of diagnosis, and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood (30 mL) collected in EDTA-coated tubes by centrifugation on Ficoll-Paque and were processed immediately. Forward scatter (FSC) and sideward scatter (SSC) on a linear scale were used for gating live cell populations. Then, CD4⁺ and CD8⁺ T cells were analyzed by flow cytometry. Anti-CD4-FITC and anti-CD8-PE were purchased from eBioscience (San Diego, CA, USA). Myeloid-derived suppressor cells (MDSCs) were divided into 2 categories: granulocytic MDSC (G-MDSC) and monocytic MDSC (M-MDSC). For G-MDSC, cells labeled with anti-HLA-DR-PerCP (BD BioSciences, San Jose, CA, USA) and anti-Lineage Cocktail 1 (Lin1)-FITC (BD BioSciences) were gated and then identified using rat anti-mouse CD11b-APC-Cy™7 (BD BioSciences) and mouse anti-human CD33-V450 (BD BioSciences) antibodies. The frequency of G-MDSC immunophenotyped as the HLA-DR^-Lin^-CD11b⁺CD33⁺ population was quantitated as a percentage of PBMC. For M-MDSC, cells labeled with anti-HLA-DR-PerCP (BD BioSciences) and anti-human CD14-APC antibodies (eBioscience) were gated. The frequency of M-MDSC immunophenotyped as the HLA-DR^-CD14⁺ population was quantitated as a percentage of PBMC. Flow cytometry was performed using a FACSCalibur™ (BD Biosciences).