Anti cd15 clone w6d3

Murine neutrophils were isolated following harvest of bone marrow from 8-week-old naïve B6 or PD-L1 KO mice. In brief, skin, muscle and fat were removed from femurs and tibias. Bone marrow was then flushed using a 27g needle and RPMI (Gibco). Following isolation of pure neutrophils, cells were then treated with 250-500 nM phorbol-12-myristate-13-acetale (PMA, Sigma-Aldrich) for 4 hours and then spun at 480xg for 5 minutes. The pellet was discarded and supernatant containing NET chromatin was spun at 18,000xg for 10 minutes. Quantification of NETs was analyzed using a Nano Drop (Thermo Fisher Scientific). In-vivo NETs quantification was analyzed using myeloperoxidase (MPO) (Roche) associated with DNA ELISA (Roche). For the human studies, fresh peripheral blood obtained from healthy donors or patients was stained with anti-CD15 (clone W6D3, BioLegend), anti-CD16 (clone 3G8, BioLegend), anti-citrullinated histone H3 (Abcam), anti-PD-L1 (clone 29E.2A3, Biolegend) and specific secondary anti-antibodies (Thermo Fisher Scientific).

To examine PMN-MDSC, two-hundred thousand PBMC were stained with antibodies, comprising anti-CD66b (clone G10F5) conjugated to fluorescein isothiocyanate, anti-CD14 (clone MφP9) conjugated to allophycocyanin (both from BD Biosciences, San Jose, CA), CD11b (clone LM2) conjugated to phycoerythrin and anti-CD15 (clone W6D3) conjugated to peridinin chlorophyll protein complex (PerCP) (both from Biolegend, San Diego, CA). To provide insight into immunosuppressive function, we stained one million PBMC with anti-CD4 (clone RPA-T4) conjugated to PerCP to count CD4+ T cells in the same samples. Stained cells were acquired on a portable BD Accuri C6 flow cytometer (BD Biosciences).