Isolation of Murine Bone Marrow Neutrophils

Bone marrow–derived neutrophils were isolated from C57BL/6 mice (7 wk old, males, n = 6) as previously described (62 (link), 63 (link)). Briefly, mice were euthanized according to the approved procedure by the Hebrew University of Jerusalem Institutional Animal Care and Use Committee (IACUC). Euthanized mice were disinfected and dissected to isolate the femur. Muscles on the femur were gently removed with scalpel and scissors while avoiding damage to the head of the femur. The bones were rinsed in 70% ethanol and washed with cold Dulbecco’s phosphate-buffered saline (Biological Industries). The epiphyses of the long bones were cut, and bone marrow cells were isolated by scraping the inner lining of the shaft and then flushing the shaft with PBS using a 25-gauge needle into a 50-mL tube fitted with a 100-μm filter. Bone marrow cells were pelleted by centrifuging at 1,500 rpm for 5 min (Eppendorf centrifuge R5810). Red blood cells were lysed by resuspending the cell pellet in 1 mL of red blood cell lysis solution (150 mM NH₄Cl, 10 mM NH₄HCO₃, and 2 nM EDTA) for 5 min at room temperature and then neutralized with 9 mL PBS. Cells were centrifuged and resuspended in RPMI-1640 (Sigma) supplemented with 10% FBS. The yield was determined by counting the cells with a hemocytometer. Lipopolysaccharide (LPS; 2 µg/mL, Thermo Scientific) was added to activate the bone marrow neutrophils.