Gr 1 fitc

Flow assisted cell sorting (FACS) was performed as described elsewhere (16 (link)) with the following conjugated antibodies; Mac1 PE, Gr1 FITC, B220 APC, B220 FITC, CD19 PE, surface IgM PE, CD43 FITC, CD3 PE, CD4 APC, CD8 FITC, CD71 PE, Ter119 FITC, Sca-1 PE, cKit FITC, cKit APC-Cy7, IL7Ra PECy7, CD34 FITC, CD16/32 PE (eBiosciences or BD Biosciences). StemSep Mouse Hematopoietic Progenitor Kit Lineage Positive antibody cocktail (Stem Cell Technologies, Vancouver, Canada) was used to detect lineage positive bone marrow cells, stained as previously described (16 (link)). Apoptosis assays were conducted using AnnexinV-FITC Apoptosis Detection Kit I following the manufacturer’s recommended protocol. IHC and immunoblotting were performed as previously described (49 (link)). Formalin-fixed paraffin embedded sections were stained with hematoxylin and eosin (H&E), myeloperoxidase (MPO) (A0398, DAKO), CD3 (MCA1477, AbD Serotec), B220 (553086, BD), Ter119 (116201, BioLegend). Stained slides were scanned and imaged as described elsewhere (49 (link)). For immunoblots, primary antibodies used were anti-V5 (ab9116, Abcam or R960-25, Life Technologies), and beta-actin (Cell Signaling Technology). Protein was visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific, Illinois, USA) and Amersham Hyperfilm ECL (GE Healthcare Ltd, Buckinghamshire, UK).

BALB/c mice were inoculated in the abdominal mammary gland with 4T1 or 4T1/IL1β tumor cells, and MDSC were harvested from the mice as described by Chornoguz et al. [9 ]. Briefly, mice with primary 4T1 or 4T1/IL1β tumors of ~7–10 mm in diameter and established metastatic disease were bled from the submandibular vein into heparinized tubes. Red blood cells were removed by lysis and the remaining leukocytes were used immediately or frozen at –80C until used. The percent of MDSC in the ex vivo leukocyte population was determined by immunofluorescence and flow cytometry using the fluorescent antibodies Gr1-FITC and CD11b-PE (eBioscience, Inc., San Diego, CA) as described [9 ]. Individual biological samples consisted of MDSC pooled from two to three individual mice and consisted of >90% Gr1⁺ CD11b⁺ cells (see Fig. 1A).

Tumor-infiltrating immune cells were incubated with Fc-blocking reagent (anti CD16/CD32, BD Biosciences), followed by CD11b-APC (M1/70, BD Biosciences), Gr1-FITC (RB6–8C5, BD Biosciences), CD11c-APC (HL3, BD Biosciences), MHC II-FITC (I-Ab, AF6–120.1, BD Biosciences), CD3 -APC (145–2C11, eBioscience), CD4-FITC (GK1.5, eBioscience), CD8a-APC (53–6.7, eBioscience) along with isotype-matched control. In vitro cultures dendritic cells (DCs) were stained with CD11b-APC, Gr1-FITC, CD11c-APC, MHC II-FITC, CD80-FITC (16–10A1, eBioscience) and CD86-FITC (GL1, eBioscience).