Anti ly 6g rb6 8c5

To obtain mouse neutrophils, BM cells were isolated from the femurs and tibias of C57BL/6 J WT, KO, and mutant mice. The BM cells were incubated with Fc blocker (93; Biolegend) and stained with biotinylated anti-Ly-6G (RB6-8C5; Biolegend) antibodies. They were then incubated with anti-biotin-microbeads (Miltenyi Biotech). Ly-6G^high cells were enriched using magnetic sorting. The purity of the isolated neutrophils was more than 95% when assessed using flow cytometry (FACSverse; BD).
To obtain human neutrophils, peripheral blood was collected from healthy adult volunteers using heparin. Red blood cells were removed using HetaSep^TM (STEMCELL Technologies) sedimentation according to the manufacturer’s protocol. Then, the cells were washed twice with RPMI 1640 medium and further fractionated on a discontinuous Percoll PLUS (GE-healthcare) gradient that consisted of layers with densities of 75%, 65%, and 55%. After the mixture was centrifuged for  30 min at 500 × g, the interface between the 65% and 75% layers was collected and washed twice with RPMI 1640 medium. All procedures were conducted at room temperature. The preparations contained more than 95% of CD15⁺CD16⁺Siglec-8⁻ neutrophils according to flow cytometric analysis. Cell viability was >98% according to trypan blue exclusion assays.

The absolute number of neutrophils were determined in freshly harvested blood and peritoneal lavage of mice. Briefly, 100 μL of blood or peritoneal lavage was stained for neutrophils and the absolute counting was performed on a flow cytometer using a bead counting method (AccuCheck, Thermo Scientific, Waltham, MA, USA) as we have previously described [12 (link),16 (link)]. Antibodies used to identify neutrophils included anti-Mac-1 (M1/70), anti-F4/80 (BM8) and anti-Ly-6G (RB6-8C5) (BioLegend, San Diego, CA, USA). The appropriate isotype-matched negative control antibodies were purchased from the same source. Bacterial counts were determined by plating 10-fold serial dilutions of blood or peritoneal lavage fluid on brain heart infusion agar plate (Becton Dickinson, Sparks, MD, USA), as previously described [16 (link)].