Isolation and Characterization of Murine Bone Marrow and Peritoneal Neutrophils

Mature murine bone marrow PMNs were isolated from mouse femurs and tibias by discontinuous Percoll gradient centrifugation as previously reported.^{19 (link)} This preparation was then sorted by a FACSAriaII (Becton Dickinson, Franklin Lakes, NJ) based on live (7AAD^neg), Ly6G⁺, and F4/80^{6 (link)} to yield a population that was >95% pure with 0% F4/80⁺ cells (Figure 7A). Peritoneal PMNs were harvested by peritoneal lavage 4h after i.p. Brewer's thioglycollate (2 ml, 4%). Peritoneal exudate macrophages were harvested by peritoneal lavage 96h after i.p. Brewer's thioglycollate and then plated for 24h before stimulation. Macrophages were stimulated with media, Pam3CSK4 (Invivogen, San Diego, CA; 100ng/ml), Poly I:C (Invivogen, 10μg/ml), LPS (List Biologicals, Campbell, CA; 10ng/ml), or CpG oligodeoxynucleotide (Invivogen; 10Qg /ml) for 24h and cell supernatants harvested for cytokine analysis.