Phrodo staphylococcus aureus bioparticles

Splenocytes from 60 hpi mice were isolated and enriched for myeloid cells by FACS staining with PE-conjugated antibodies specific for CD90.2, IgM, ad NK1.1 then incubating with α-PE beads (Miltenyi Biotec) to deplete lymphocytes as described above. The flow-through from the LS magnetic column (Miltenyi Biotec) was collected and 5 x 10⁵ myeloid cells were plated in a 24-well plate (Gibco) and allowed to adhere for at least 3 hrs. pHrodo Staphylococcus aureus Bioparticles (ThermoFisher) were reconstituted as per manufacturer’s instruction in Phagocytosis Buffer (HBSS without cations, 20 mM HEPES, 5 mM EDTA, pH 7.4) to a concentration of 1 mg/mL. The Bioparticles were added to the myeloid cells at a concentration of 30 Bioparticles per cell and incubated at 37°C (experimental) or 4°C (control) for 30 min. Phagocytosis was stopped by the addition of ice-cold Phagocytosis Buffer and the cells were placed on ice. Samples were washed twice in ice-cold Phagocytosis Buffer and once in ice-cold FACS Buffer and then fixed in 1% PFA for 30 min in the dark at room temperature before being stained for flow cytometric analysis.