0.22 μm pore size filters

P. gingivalis 33277 was grown from frozen stocks in TSB (trypticase soy broth) or on TSB blood agar plates supplemented with yeast extract (1 mg/mL), hemin (5 μg /mL), and menadione (1 μg/mL), and incubated at 37°C in an anaerobic chamber (85% N2, 10% H2, 5% CO2). P. gingivalis vesicles were prepared as previously described (Furuta et al., 2009 (link)). Briefly, P. gingivalis was grown to the late exponential phase and growth media were collected by centrifugation at 10,000 × g for 15 min at 4°C and filtered through 0.22-μm-pore-size filters (Cell Treat, MA, USA) to remove residual bacteria. Vesicles were collected by ultracentrifugation at 126,000 × g for 2 h at 4°C and resuspended in phosphate-buffered saline (PBS) containing 10% glycerol.

P. gingivalis strains are listed in Table 1, They were grown from frozen stocks in TSB (trypticase soy broth) or on TSB blood agar plates supplemented with yeast extract (1mg/ml), hemin (5 μg /ml) and menadione (1μg/mL), and incubated at 37°C in an anaerobic chamber (85% N2, 10% H2, 5% CO2). Streptococcus strains were grown in Trypticase Peptone Broth (TPB) supplemented with 0.5% glucose at 37°C under aerobic conditions. P. gingivalis vesicles were prepared as previously described [16 (link)]. Briefly, P. gingivalis was grown to the late exponential phase and growth media were collected by centrifugation at 10,000 × g for 15 min at 4°C and filtered through a 0.22-μm-pore-size filters (CellTreat) to remove residual bacteria. Vesicles were collected by ultracentrifugation at 126,000 x g for 2 h at 4°C and resuspended in phosphate-buffered saline (PBS) containing 10% glycerol. Since quantifying vesicles by their protein or lipid content in weight represents the most common way to normalize data [1 (link)], for these studies proteins were extracted from vesicles using a BugBuster Protein Extraction Reagent (Novagen) and protein concentrations were determined with a Bio-Rad Protein Assay Kit (Bio-Rad).

P. gingivalis 33277 was grown from frozen stocks in trypticase soy broth (TSB) or on TSB blood agar plates supplemented with yeast extract (1 mg/mL), hemin (5 μg/mL) and menadione (1 μg/mL), and incubated at 37 °C in an anaerobic chamber (85% N2, 10% H2, 5% CO2). P. gingivalis vesicles were prepared as previously described^{46 (link)}. Briefly, P. gingivalis was grown to the late exponential phase, and growth media were collected by centrifugation at 10,000 × g for 15 minutes at 4 °C and filtered through 0.22-μm-pore-size filters (Cell Treat, MA) to remove residual bacteria. Vesicles were collected by ultracentrifugation at 126,000 × g for 2 h at 4 °C and resuspended in phosphate-buffered saline (PBS) containing 10% glycerol. OMVs were quantitated using both protein and lipid assays. Proteins and lipids were extracted from vesicles in PBS using a BugBuster® Protein Extraction Reagent (Novagen, MA).