Biotin coupled mouse anti gr1 antibody clone rb6 8c5

Gr1⁺CD11b⁺ cells were isolated from the bone marrow using magnetically assisted cell sorting according to the manufacturer’s protocol (Miltenyi Biotech, Auburn, CA). The bone marrow cells were flushed out of the femurs with RPMI-1640 medium (without serum) under aseptic conditions (Brudecki et al., 2012b (link)). A single cell suspension of the bone marrow was made by pipetting up and down and filtering through a 70-μm nylon strainer, followed by incubation with erythrocyte lysis buffer. After washing, total Gr1⁺CD11b⁺ cells were purified by subjecting the single cell suspension to positive selection of the Gr1⁺CD11b⁺ cells by incubating with biotin-coupled mouse anti-Gr1 antibody (Clone RB6-8C5; eBioscience, San Diego, CA) for 15 min at 4°C. Cells were then incubated with anti-biotin magnetic beads for 20 min at 4°C and subsequently passed over a MS column. Purified Gr1⁺CD11b⁺ cells were then washed and resuspended in sterile saline. The cell purity was determined by flow cytometry. Typically ~90% Gr1⁺CD11b⁺ cells were obtained by this procedure.
Gr1⁺CD11b⁺ cells were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine (all from Hyclone Laboratories, Logan, UT), and 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) at 37°C and 5% CO₂.

Bone marrow Gr1⁺CD11b⁺ cells were isolated using magnetic beads according to the manufacturer’s protocol (Miltenyi Biotech, Auburn, CA). Briefly, the bone marrow was flushed out of the femurs with RPMI-1640 medium (without serum) under aseptic conditions. A single cell suspension was made by pipetting up and down and filtering through a 70-μm nylon strainer, followed by incubation with erythrocyte lysis buffer and washing. The cell suspension was subjected to a positive selection of the Gr1⁺ cells by incubating with biotin-coupled mouse antiGr1 antibody (Clone RB6–8C5; eBioscience, San Diego, CA) for 15 min at 4°C. Cells were then incubated with anti-biotin magnetic beads for 20 min at 4°C and subsequently passed over an MS column. The cell purity was more than 90% as determined by flow cytometry.
Gr1⁺CD11b⁺ cells were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine (all from Hyclone Laboratories, Logan, UT), and 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) at 37°C and 5% CO₂. In some experiments, cells were stimulated for 6 hr with recombinant murine IL-10.