Twenty-three–week–old WT C57BL/6 MPO+/+ (Taconic Biosciences, Rensselaer, NY) were intraperitoneally injected with 200 μg of Gr-1 antibody (RB6-8C5, Bio X Cell, West Lebanon, NH, USA) per mouse every 3 days (39) for 3 weeks. Peripheral blood was first drawn using an intracardiac puncture with a 25G needle in Gr-1–treated animals and age-matched MPO+/+ control animals under anesthesia. The spleen was harvested and a single-cell suspension of cells was created using a 70-μm strainer. Bone marrow was harvested from the femur and tibia using the protocol described above. To quantify CD11b+Ly6G+ neutrophils by flow cytometry, 1 × 106 of the isolated blood, bone marrow, and spleen cells from αGr-1–treated and control animals were labeled with 5 μl of CD11b-FITC (Miltenyi Biotec, Auburn, CA) and 5 μl of anti–Ly6G-APC (Miltenyi Biotec, Auburn, CA) in 45 μl of flow buffer [PBS (pH 7), 0.5% BSA, and 2 mM EDTA]. Flow cytometry analysis was done at the South Campus Flow Cytometry and Cell Sorting Core at UT MD Anderson Cancer Center.

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