Trustain fcx

Non-specific antibody binding was blocked by incubating cells with TruStain FcX (eBioscience, San Diego, CA, USA) on ice for 15 minutes. The cells were subsequently incubated with monoclonal antibodies (Table 1) on ice for 30 minutes. The monoclonal antibodies included anti-human CD45 (HI30, BioLegend, San Diego, CA, USA) for whole leukocytes, anti-human CD3 (HIT3a, BD Pharmingen, San Jose, CA, USA) for T cells, anti-human CD14 (HCD14, M5E2, BioLegend, San Diego, CA, USA) for macrophages, anti-human CD19 (HIB19, BioLegend, San Diego, CA, USA) for B cells, anti-human CD11c (3.9, BioLegend, San Diego, CA, USA) for dendritic cells, anti-human CD16 (3G8, BioLegend, San Diego, CA, USA) for granulocytes including neutrophils and eosinophils, and anti-human HLA-DR (L243, BioLegend, LN3, eBioscience, San Diego, CA, USA) for dendritic cells and B cells. Flow cytometry was performed using a FACS Canto II (BD Biosciences, San Jose, CA, USA) and data were analyzed using Flow Jo ver. 9.3.2 (Treestar, Inc., San Carlos, CA, USA) (Fig 1).

Tumor tissues of mice were obtained and single-cell suspensions were prepared. The cells were blocked with anti-mouse CD16/32 (TruStain fcX™, USA) and identified with anti-mouse CD3, CD4, CD8, and CD45 antibodies (eBioscience). Then single-cell suspension samples were run on a BD FACSVerse™ (BD Biosciences, USA) and analyzed using FlowJo software (TreeStar, USA).