Phrodo staphylococcus aureus bioparticles conjugate

pHrodo Staphylococcus aureus BioParticles conjugate (Invitrogen) was used to determine in vitro phagocytic capacity (S. D. Brown, Gauthier, & Brown, 2009 (link)) of MH-S cells or mAM (1.2 × 10⁵ cells each). As reported (S. M. Yeligar, Mehta, et al., 2016 (link)), cells were incubated with 1 × 10⁶ particles of pH-sensitive pHrodo S. aureus BioParticles conjugate for 2 h, followed by fixation with 4% paraformaldehyde. Phagocytosis of bacteria was analyzed using an Olympus confocal microscope, and cells from 10 fields per experimental condition were assessed using quantitative digital fluorescence imaging software (Olympus FluoView 300, Version 4.3). To measure S. aureus internalization, argon/krypton laser confocal microscopy was performed at 50% of cell depth using identical background and gain settings. All cells with internalized bacteria were considered positive for phagocytosis. Phagocytic index (calculated as the percentage of cells positive for phagocytosis multiplied by the RFU of S. aureus per cell) was used to quantify phagocytosis and is expressed as mean ± SEM, relative to average control values.