Mouse neutrophil isolation kit

Mice were euthanized with 2% isoflurane followed by cervical dislocation, and the femur and the tibia from both hind legs were removed and freed of soft tissue attachments. Then, the distal tip of each bone was removed, and marrow cavities were flushed in RPMI 1640 media with 10% fetal bovine serum (FBS). Neutrophils were isolated by centrifugation over a discontinuous Percoll gradient consisting of 36% (v/v) and 72% (v/v) Percoll in PBS at 700g for 20 min at 25°C. Neutrophils were recovered at the interface of the 36 and 72% fractions with >85% purity and viability as determined by flow cytometry (6 (link)). For in vitro cytokine and chemotaxis analysis, bone marrow neutrophils were purified with a mouse neutrophil isolation kit (STEMCELL) following the manufacturer’s protocol. This population consisted of more than 90% neutrophils as measured by flow cytometry (fig. S7).