Phagocytosis assay was performed as previously reported with some modifications (Daigneault et al., 2010 (link)). J774.1 or THP-1 macrophages were seeded at 1.0 × 105 cells into each well of a 96-well plate and incubated with fourfold serial dilutions of kinase inhibitors (from 128 to 0.03 μM) for 2 h at 37°C. Then, red fluorescent latex beads with a mean diameter of 2 μm (L3030; Sigma-Aldrich) were opsonized by 10% human serum (Cosmo Bio) for 30 min at 37°C. The opsonized beads were added to cells at an MOI of 10. The cells were then incubated for 2 h with different concentrations of inhibitors. Cells were washed twice with PBS, and red fluorescence (excitation, 535 nm/emission, 595 nm) was analyzed by a FACS Calibur flow cytometer (BD). We used 10 μM of cytochalasin D (Sigma-Aldrich) as a control, which completely inhibits actin polymerization (Cooper, 1987 (link)).
The fluorescence was compared with controls in 5,000 events. The phagocytosis rate (%) was calculated according to the following formula: phagocytosis (%) = 100 × [fluorescent cells (sample—negative control) / fluorescent cells (positive control – negative control)]. Positive and negative controls were the addition of fluorescent beads and DMSO alone, respectively.
The fluorescence was compared with controls in 5,000 events. The phagocytosis rate (%) was calculated according to the following formula: phagocytosis (%) = 100 × [fluorescent cells (sample—negative control) / fluorescent cells (positive control – negative control)]. Positive and negative controls were the addition of fluorescent beads and DMSO alone, respectively.