Macs mouse anti ly 6g microbead kit

Mice were sacrificed and the tibia and femur were extracted and cleaned from excess flesh. The ends of each tibia and femur were clipped with dissecting scissors and the bone marrow cells were flushed with ice cold PBS^−/− using a syringe with 27-G needle. The pooled bone marrow was then separated by gentle pipetting, followed by filtration through a sterile 70-μm nylon cell strainer to remove cell clumps and bone particles. The filtrated cells were centrifuged for 5 min at 1,500 rpm and resuspended in complete DMEM. To prepare Bone Marrow Macrophages (BMM), cells were plated 5 × 10⁶ cells/ plate and cultured in complete DMEM supplemented with 15% L cell supernatant as a source of M-CSF, and 15% horse serum. Media was replaced after 3 days and cells were collected for use after 1 week. To stimulate, BMM were plated in triplicate at 400,000 cells/well, in 96-well flat bottom plates in complete DMEM. Bone marrow neutrophils were prepared by negative or positive selection from pooled bone marrow cells. Negative selection was performed using the EASYSEP® magnet cell selection system (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer's instructions. Positive selection was performed using the MACS® mouse Anti-Ly-6G MicroBead Kit (Miltenyi Biotec Inc., CA, USA) according to manufacturer's instructions.

The femurs and tibias were isolated and the soft tissue was removed. The ends of the bones were opened and the bone marrow were flushed out with sterile PBS. Afterward, cells were isolated as described elsewhere (Swamydas et al., 2015 (link)). Briefly, the cells were centrifuged (300 ×g, 10 min, 4°C), resuspended in 2 ml of MACS buffer and passed through a 30 μm cell strainer to remove any debris. The PMNs were separated by positive selection using the MACS mouse Anti-Ly-6G Microbead Kit (Miltenyi Biotech, Bergisch Gladbach, Germany) following the manufacturer’s instructions. After positive selection the neutrophils were resuspended in RPMI1640 with 2% FCS, 1% Glu and cells were stimulated immediately following isolation.

Cells were flushed out from the femurs and tibiae with sterile PBS, and PMNs were isolated via positive selection using the MACS mouse Anti-Ly-6G Microbead kit (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer's instructions. The isolated neutrophils were resuspended in RPMI 1640 with 2% FCS, 1% Glu and stimulated immediately.