Isolation of Neutrophils from Bone Marrow

Bone marrow fractionation was performed using a modification of the density gradient centrifugation method previously described⁴⁹ . Briefly, bone marrow was flushed from the femora and tibiae with 10 ml of sterile PBS and passed through an 18-gauge needle to disrupt larger bone marrow clumps. Cells were centrifuged at 300 × g for 7 min at 4 °C. Red blood cells were lysed by resuspending a cell pellet in 0.2% NaCl for 20 s followed by the addition of 1.6% NaCl. Cells were centrifuged at 300 × g for 7 min at 4 °C, washed with 2 mM EDTA in PBS and filtered through a 40 μm filter. Using a 15 ml conical tube, 3 ml of Histopaque 1119 (density 1.119 g ml⁻¹, Sigma-Aldrich) was overlaid with 3 ml of Histopaque 1077 (density 1.077 g ml⁻¹, Sigma-Aldrich). Bone marrow cells were resuspended in 1 ml of ice-cold PBS and laid over the Histopaque gradient. Samples were centrifuged for 30 min at 700 × g at 25 °C without a break. Neutrophils were collected at the interface of the Histopaque 1119 and Histopaque 1077 layers and then washed twice with PBS and used for further experiments. The composition of the cell population was confirmed using microscopy to have neutrophil morphology as determined by Giemsa staining.