Fusobacterium nucleatum (JCM8532, ATCC10953, JCM11024, and ATCC23726) and Porphyromonas gingivalis (ATCC33277) were cultured by brain heart infusion (BHI, Becton Dickinson, Sparks, MD, USA) supplemented with 5 µg/mL hemin and 0.5 µg/mL menadione anaerobically at 37 °C. Streptococcus mutans (UA159) was cultured by BHI anaerobically at 37 °C. Pseudomonas aeruginosa (PAO1) and Escherichia coli (ATCC23511) were grown using lennox L broth (LB, Thermo Fisher Scientific, MA, USA) with shaking at 37 °C. All bacteria were cultured to the absorbance at OD590 = 1.0 and used for following experiments.

For F. nucleatum inactivation, the bacterial cells were heat-inactivated at 100 °C for 15 min. Killing was confirmed by absence of growth on sheep blood agar plate (Becton Dickinson, Sparks, MD, USA). Other bacteria, P. gingivalis, S. mutans, P. aeruginosa, and E. coli were also heat-inactivated at 100 °C for 15 min.

To analyze the presence of bacteria, F. nucleatum were stained with CytoTell UltraGreen (AAT Bioquest, CA, USA) in accordance with the manufacturer’s instructions. Briefly, the bacterial cells were treated with CytoTell UltraGreen for 15 min at 37 °C, and then treated to HOC621 cells at the concentration of 100 MOI for 3 h. The cells were fixed with 4% formalin for 30 min and then permeabilized with 0.1% Triton X-100 in PBS for 2 min on ice, followed by Hoechst 33,342 for 30 min. The samples were mounted and observed by a Nikon A1 laser fluorescence confocal microscopy (A1 R HD25, Nikon, Tokyo). Images were acquired with the NIS-Elements software (Nikon, Tokyo, Japan).

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