Pg lps

The cells were seeded in 24 well plates at a density of 2×10⁴ cells/well and incubated at 37°C in a humidified atmosphere of 5% CO₂ during 18 hours in DMEM 1% FBS. After overnight adhesion, DMEM containing 1 μg/mL of Pg LPS (Invivogen, San Diego, CA, USA) or Ec LPS (Invivogen) was added to the wells for 24 or 48 h. Non-stimulated cells were used as controls. Both Pg LPS and Ec LPS were ultrapure and purchased from Invivogen.

Human neuroblastoma SK-N-SH cells overexpressing wild-type APP695 (SK-N-SH APPwt) were a gift from Dr. Dennis Selkoe (Boston, MA, USA). SK-N-SH APPwt cells were grown in DMEM, plus10% fetal bovine serum (Hyclone, Los Angeles, CA, USA), 100 U/ml penicillin/streptomycin. Also, cells were supplemented with 200 μg/ml G418. Cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2 and passed every 2–4 days based on 85% confluence. P.g-LPS was obtained from Invivo Gen (San Diego, CA, USA), and applied to the supernatant of SK-N-SH APPwt cells with the final concentration 1 μg/ml for a total of 7 days to mimic CP in vitro (P.g-LPS was incubated for 4 days initially. At confluence, SK-N-SH APPwt cells were passaged and new P.g-LPS were applied to the supernatant immediately and incubated for another 3 days). For Cathepsin B inhibition, a final concentration of 75 μM CA-074 methyl ester (Sigma-Aldrich, USA) was applied to the supernatant of SK-N-SH APPwt cells 1 h before P.g-LPS. For Cathepsin B activation, IL-6 (10 ng/ml, Genscript, Z03134), IL-1β(100 pg/ml, Genscript, Z02978), TNF-α (10 ng/ml, Genscript, Z01001) and recombinant Human CRP protein (1 mg/L, Abcam ab171471) were applied to SK-N-SH APPwt cells for 24 h. For TNF-α inhibition, pomalidomide (2 μM, Selleck, S1567) was applied to P.g-LPS treated SK-N-SH APPwt cells 24 h before cell harvest.

Since quantifying vesicles by their protein or lipid content in weight represents the most common way to normalize data (Kulp and Kuehn, 2010 (link)), we quantitated OMVs using both protein and lipid assays. Proteins and lipids were extracted from vesicles in PBS using a BugBuster® Protein Extraction Reagent (Novagen, MA, USA). The quantity of OMV lipid was assessed using the fluorescent lipophilic dye FM4-64 as described (Macdonald and Kuehn, 2013 (link)), and was quantitated by titration of P. gingivalis lipopolysaccharide (LPS-PG, InvivoGen, San Diego, California). Protein concentrations were determined with a Bio-Rad Protein Assay Kit (Bio-Rad, CA, USA). Results revealed that 1 × 10⁶P. gingivalis cells is equivalent to 100 ng protein or 3.6 μg lipid of vesicles. Thus, for in vitro infection experiments, 1 × 10⁵ host cells were exposed to 1 × 10⁶P. gingivalis cells or vesicles with 100 ng protein.