Poly l lysine coated cover glass

Three bacterial strains (S. felis ATCC 49168, S. pseudintermedius ATCC 49051, and S. schleiferi ATCC 43808) were grown overnight in lysogeny broth (LB) at 37°C. Each bacterial culture (10 µl) was inoculated in LB containing different α-MG concentrations (1/4×, 1/2×, 1×, 2×, and 4× MICs) in six-well plates with poly-L-lysine-coated cover glass (Sigma-Aldrich), and the plates were incubated at 37°C for 2 h. The cover glasses were removed from the plates and washed twice with phosphate-buffered saline (PBS). The samples were serially dehydrated with 20%, 50%, and 70% ethanol for 10 min, and 100% ethanol for 30 min. The samples were coated with platinum, and ultrastructural changes of bacteria were observed using FE-SEM (Hitachi SU-8220; Hitachi, Japan).

The mouse RAW 264.7 macrophage cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. RAW 264.7 cells and mouse peritoneal cells were cultured overnight on poly-L-lysine-coated cover glass (Sigma) in 12-well plates (Nunc, Roskilde, Denmark). FITC-labeled S. aureus MW2 was incubated with PBS or antibodies for 1 h and then the bacteria were added to the 12-well plates. After 1 h incubation, the cells were fixed with 4% paraformaldehyde (Affymetrix, Santa Clara, CA, USA), washed with PBS, and stained with Hoechst No. 33258 (Sigma-Aldrich) at room temperature to identify cell nuclei. The mounted cells were analyzed using a LSM 710 laser scanning microscope (Carl Zeiss, Oberkochen, Germany). The phagocytic index was measured by counting the number of FITC-labeled S. aureus MW2 cells phagocytosed by RAW 264.7 cells and mouse peritoneal cells as previously described^{47 (link)}.