Amicon ultra10 filters

Differentiated THP-1 cells seeded in 12-well plates (1.0 × 10⁶ cells/well) were primed with 100 ng/mL LPS for 4 h, followed by treatment with the peptide solution or nigericin for 3 h. Culture supernatants were collected and concentrated using AMICON Ultra-10 filters (Millipore). After removal of the supernatant, the cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich). The specimens were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Immunoblotting was essentially performed as described previously [13 (link)]. The membranes were incubated with primary antibodies specific for IL-1β (D3U3E, Cell Signaling Technology), cleaved IL-1β (D3A3Z, Cell Signaling Technology), or ɑ/β-tubulin (Cell Signaling Technology). After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized using the ECL Advance western blotting substrate (Thermo Fisher Scientific) and a Light-Capture II imaging analyzer (ATTO).

CPS was extracted as described previously (20 (link)). Briefly, bacteria were grown to mid-log phase in 30 mL THB at 37°C. The cell densities were measured based on OD₆₀₀. Then, the cells were pelleted by centrifugation at 3,500 × g for 15 min, washed twice with PBS, suspended in 0.8 M NaOH, and incubated for 48 h at 37°C. After being neutralized with HCl, the samples were centrifuged at 10,000 × g for 10 min and filtered to remove the insoluble material. The supernatant was concentrated using Amicon Ultra10 filters (Millipore, Bedford, MA, USA). After being washed twice with distilled water, the extracted polysaccharide was collected from the membrane by resuspension in water. The concentrations of sialic acid were determined by a sialic acid assay kit (Jiancheng, Nanjing, China) according to the manufacturer’s instructions.

CPS was extracted as described previously (20 (link)). Briefly, bacteria were grown to mid-log phase in 30 mL THB at 37°C. The cell densities were measured based on OD₆₀₀. Then, the cells were pelleted by centrifugation at 3,500 × g for 15 min, washed twice with PBS, suspended in 0.8 M NaOH, and incubated for 48 h at 37°C. After being neutralized with HCl, the samples were centrifuged at 10,000 × g for 10 min and filtered to remove the insoluble material. The supernatant was concentrated using Amicon Ultra10 filters (Millipore, Bedford, MA, USA). After being washed twice with distilled water, the extracted polysaccharide was collected from the membrane by resuspension in water. The concentrations of sialic acid were determined by a sialic acid assay kit (Jiancheng, Nanjing, China) according to the manufacturer’s instructions.