Pe ly6g antibody

Bone marrow-derived neutrophils were isolated from mice using the following protocol adapted from Swamydas et al. 6 . Bone marrow cells were obtained from mice by flushing the contents of the tibia and femur with complete RPMI medium supplemented with 2 mM EDTA using a 25-gauge needle. The cell suspension was run through a 100 µM cell strainer. The cells were pelleted at 430xg for 7 minutes at 4°C. Red blood cells were lysed by washing the cells with 20 mL of 0.2% NaCl for 20 seconds followed by the addition of 20 mL of 1.6% NaCl. After washing with PBS, the bone marrow cells were resuspended in ice-cold PBS and layered on the top of a Histopaque 1119/1077 (Sigma) gradient in a 15 mL Falcon tube. After centrifugation at 2000 rpm at room temperature for 30 minutes (without the break), the neutrophils were isolated by collecting the cells at the interface of the two Histopaque layers. The neutrophils were then washed twice with complete RPMI medium. Neutrophil viability and purity were assessed via trypan blue staining and flow cytometric analysis of Ly6G surface expression by using a PE-Ly6G antibody (Biolegend).