Cultivation of P. gingivalis Strains

P. gingivalis ATCC33277 and Lys gingipain mutant P. gingivalis KDP129 were used. P. gingivalis was maintained on blood agar plate containing 40 mg/ml trypto-soya agar (Nissui Pharmaceutical, Tokyo, Japan), 5 mg/ml brain heart infusion (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), 1 g/ml cysteine (Wako Pure Chemical Industries, Osaka, Japan), 5 μg/ml hemin (Sigma-Aldrich, St. Louis, MO, USA), 1 μg/ml menadione (Sigma-Aldrich), 5% defibrinated sheep blood (Nippon Bio-test laboratories, Tokyo, Japan) in Bactron anaerobic chamber (Shel Lab, Cornelius, OR, USA) with 10% CO₂, 10% H₂, 80% N₂^{15 (link)}. KDP129 was maintained on blood agar plate with 20 μg/ml chloramphenicol (Wako Pure Chemical Industries). P. gingivalis was grown in enriched BHI broth containing 37 mg/ml brain heart infusion, 2.5 mg/ml yeast extract (Becton, Dickinson and Company), 1 g/ml cysteine, 5 μg/ml hemin and 1 μg/ml menadione. KDP129 was grown on BHI broth with 20 μg/ml chloramphenicol. Before P. gingivalis was cocultured with MG6 cells and primary cultured microglia, P. gingivalis culture medium was centrifuged (6000 × g, 10 min) and the supernatant was replaced with DMEM without FBS or penicillin-streptomycin.