Lipopolysaccharides from e coli 0111 b4

Inositol hexaphosphoric acid (IP6, #P8810) and Lipopolysaccharides from E. coli 0111:B4 (LPS, #L2630) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). IP6 powder was dissolved in distilled water at 20 mg/ml with pH adjusted to 7.4. The stock solution was then stored at −20°C before being further diluted for cell culture. LPS was prepared in a phosphate buffer at 1 mg/ml and stored at −20°C before use. Dulbecco's Modified Eagle Medium (DMEM, #12430047), RPMI1640 (#A4192301), phosphate‐buffered saline solution (PBS, #70011), and fetal bovine serum (FBS, #26140), L‐glutamine (#21051024), penicillin–streptomycin (#15140148), paraformaldehyde (#R37814), and Trypsin‐EDTA solution (#R001100) were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA).

BV2 cells were seeded at 50 × 10³ cells per well in 24-well plates (Corning costar, Cat #3524) and incubated overnight to achieve 70–80% confluency. Media was replaced with a fresh 200 µl DMEM media supplemented with 10% Horse serum and 1% antibiotics (cat #15240-062, Life technologies). Unless specified, cells were incubated in triplicates with 10 µl of purified PrP^Sc or mock-purified materials for 18 hr at 37 °C and 5% CO₂ in DMEM media supplemented with 10% HS and 1X antibiotics. Primary microglia cells seeded in 24-well plates were incubated with 10 µL of purified PrP^Sc or mock-purified materials for 18 h in 200 µL cDMEM media (10% FBS, 1% antibiotic (cat #15240-062, Life technologies) at 37 °C and 5% CO₂ in a humidified chamber. Under standard conditions, the final concentrations of brain- and cell-derived PrP^Sc in the cell culture medium were estimated to be equivalent of PrP^Sc in 0.75% 22L scrapie brain homogenate or 0.15% 22L ScN2a cell lysate. Cell cultures treated with 100 ng/ml lipopolysaccharides (from E. coli 0111:B4, Sigma, cat #L3012) served as positive controls of inflammatory response. After incubation with PrP^Sc for 18 hours, supernatants and cells were collected and used for analysis of nitrite oxide, TNFα, IL6 and iNOS.