Opsonization and Phagocytosis Assays

Sheep RBCs or 3.87 μm LBs were used as target particles for macrophages in phagocytosis assays. BMDMs were incubated in serum-free DMEM at 37°C at 5% CO₂ for 1.5 h before the phagocytosis assays. Prior to opsonization, 100 μL of sRBCs (10% suspension) were washed with 500 μL of PBS and then opsonized with 2 mg/mL of anti-sheep rabbit IgG or 0.06 mg/mL of anti-sheep rabbit IgM for 1 h on a rotator at RT. The LBs were opsonized with 2 mg/mL of human IgG or human IgM. After opsonization, IgM-opsonized sRBCs/LBs were washed with 500 μL of PBS supplemented with 0.5 mM CaCl₂ and 0.5 mM MgCl₂. C5-deficient human serum (50 μL) was then added to the sRBCs/LBs and incubated in a water bath at 37°C for 30 min. The C3bi/IgM-opsonized sRBCs/LBs were then washed twice with 500 μL of supplemented PBS before being used in phagocytosis assays. Forty to Sixty microliter of opsonized particles were added to macrophages to induce an average uptake of 1–2 particles per macrophages. CR was activated in macrophages with either 100 nM PMA (19) in serum-free DMEM for 7 min or 300 U/ml GM-CSF for 1 h (23, 24) prior to the addition of C3bi-sRBCs/LBs.