The mouse RAW 264.7 macrophage cell line was purchased from the American Type Culture Collection (ATCC). The cells were cultured overnight on poly-L-lysine-coated cover glass (Sigma-Aldrich Co.) in 12-well plates (Nunc Inc.). The FITC-labeled S. aureus MW2 was treated with PBS or IgM for 1 h, and then, the bacteria were added to the plate cultured with RAW 264.7 cells. The cells were incubated for 1 h and fixed with 4% paraformaldehyde (Affymetrix Inc.). The fixed cells were washed with PBS, and then, Hoechst No. 33258 (Sigma-Aldrich Co.) was added at room temperature. The mounted cells were analyzed using a LSM 710 laser scanning microscope (Carl Zeiss Co.) previously described (18 (link)). The phagocytic index was measured by counting the number of FITC-labeled S. aureus MW2 phagocytosed by RAW 264.7 cells as previously described (18 (link)).
Additional Material and Methods are provided in the Supplementary Material.