Rb6 8c5

To determine the role of neutrophils in USA300-C2406 infection, a depletion process was employed using the antibody RB6-8C5 (BioXCell, West Lebanon, NH, USA), which mainly depletes neutrophils, at different time points in mice with a USA300-C2406. There were at least 3 mice for each group and each time point. As noted above, mice were injected with 10⁷ CFU of USA300-C2406 intradermally in the intrascapular region on day 0. Mice in the early depletion groups received an intraperitoneal (i.p.) injection of 200 µg RB6-8C5 24 h before infection, followed by injections every 48~72 h. Mice in the late depletion groups received an i.p. injection of 200 µg RB6-8C5 24 h after infection, followed by injections every 48~72 h. Mice in the control group were injected i.p. with 200 µg of rat IgG2 isotype control monoclonal antibody (BioXCell), on the same schedule with the depletion antibody RB6-8C5. All mice were carefully monitored (including obtaining weights), then euthanized on days 4, 7, and 14 post-infection. Lung, liver, and spleen were harvested and homogenized in 1 mL sterile saline, then quantitative cultures (serial dilutions and spread on TSA plates) were performed to determine bacterial load, as described above. The skin samples were processed into tissue sections and affixed to microscope slides for Gram staining, as described above.

Anti-GR1 (Bio X Cell, clone RB6-8C5) or a rat IgG2b isotype control (Bio X Cell, clone LTF2) antibodies were injected intraperitoneally from 1 to 3 days post-infection (0.25 mg per mouse and per day), in saline. The efficacy of the depletion was assessed by histology and flow cytometry (data not shown).