Pe cy5 conjugated anti mouse cd11b

Cells were dissociated from tumors according to methods described previously (29 ). Total cells dissociated from tumors were blocked with rat anti-mouse CD16/32 IgG (BD Pharmingen) and then stained with FITC conjugated anti-mouse Ly-6C (clone: HK1.4), phycoerythrin (PE) conjugated anti-mouse CD11c (clone: N418), PE-Cy5 conjugated anti-mouse CD11b (clone: M1/70), APC conjugated anti-mouse NK1.1 (clone: PK136) (all from eBioscience) and APC-Cy7 conjugated anti-mouse Ly-6G (clone: 1A8) (BioLegend). All antibodies were diluted 1:400. Data were collected from at least 200,000 single cells by FACSCanto (BD Pharmingen) and analyzed by Flowjo (Tree Star, Inc). Tumor-associated immune cells were characterized based on the expression of phenotypic markers (30 ). Dendritic cells: CD11b⁺ CD11c⁺, monocytes: CD11b⁺ CD11c⁻, monocytic myeloid derived suppresser cells (MDSCs): CD11b⁺ CD11c⁻ Ly6GLy6C^high, and granulocytic MDSCs: CD11b⁺ CD11c⁻ Ly6G⁺ Ly6C^low.

PBMCs were isolated according to methods described elsewhere (16 (link)). PBMCs were blocked using a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with FITC conjugated anti-mouse CD45.2 (clone: 104), APC-eFluor780 conjugated anti-mouse CD45.1 (clone: A20), PE conjugated anti-mouse B220 (clone: RA3–6B2), PE-Cy5 conjugated anti-mouse CD11b (clone: M1/70) (all acquired from eBioscience) and Brilliant Violet 421 conjugated anti-mouse CD3e antibodies (clone: 145–2C11; BioLegend). All antibodies were diluted 1:400. Data were collected from at least 20,000 single cells by FACSCanto (BD Pharmingen) and analyzed using FlowJo (Tree Star, Inc) without knowledge of the genotype or treatment by a single observer (C-LL).