In Vitro Longitudinal Confocal Fluorescence Microscopy and Imaging Cytometry to Quantify Neutrophil Swarming, Opsonophagocytosis, and Vital NETosis During Implant‐Associated Infection

ABSTRACT The “Race for the Surface” hypothesis remains the dogma for explaining the competition between host proteins, cells, and bacteria for implant colonization immediately post‐op. Recently, we used intravital microscopy to provide direct evidence supporting this hypothesis by demonstrating that the race for the surface in a murine implant‐associated infection (IAI) model is complete within 3 h. However, this intravital imaging is impractical for early‐stage drug development research, thus warranting a cost‐effective screening platform to mechanistically assess neutrophil‐ S. aureus interaction dynamics during the early infection period. To address this, we developed an in vitro IAI model using longitudinal confocal fluorescence microscopy (LCFM) of etched titanium implants contaminated with GFP + USA300 and cultured with tdTomato + neutrophils. This model enabled reproducible quantification of neutrophil swarming, phagocytosis, and nidus formation, which were validated with scanning electron microscopy, colony‐forming unit assays, and imaging cytometry. To demonstrate the utility of these novel platforms, we evaluated an anti‐glucosaminidase (Gmd) mAb known to mediate opsonophagocytosis of aggregated S. aureus and assessed its ability to increase total neutrophil volume and S. aureus internalization by neutrophils and cytoplasts. Collectively, these results demonstrate the utility of the LCFM and imaging cytometry platforms for defining mAb functions that enhance neutrophil and cytoplast activity to prevent IAI.