10k membrane

The medium from treated cells were collected prior to cell lysis and 500–1000 µL was concentrated in Amcion® ultra centrifugal filters with a 10K membrane (Millipore, Billerica, MA) by spinning at 14,000g for 30 min. Western blot analyses were performed on concentrated supernatants. A rabbit polyclonal HMGB1 antibody (Abcam, Cambridge, MA) was used at a dilution of 1:500. A rabbit polyclonal caspase-1 (p20) antibody (Cell Signaling Technology, Danvers, MA) was used at a dilution of 1:500. A rabbit polyclonal IL-1β antibody (Cell Signaling Technology, Danvers, MA) was used at a dilution of 1:500.

The Quantikine Human IL-1β/IL-1f2 Immunoassay (R&D Systems, Minneapolis, MN, measures predominantly mature IL-1β) was used on concentrated cell medium. Medium (500 µL) was concentrated using Amcion® ultracentrifugal filters with a 10 K membrane (Millipore, Billerica, MA) by spinning at 14,000× g for 30 min. Columns were then reversed into new collection tubes and spun for 2 min at 1,000× g. IL-1β assay was performed on the concentrated medium according to the manufacturer’s instructions. A total of 500 µL of cell supernatant was concentrated. Two hundred µL samples with assay diluents were loaded into 96 well plates pre-coated with IL-1β antibody. Values are expressed as pg/mL of IL-1β from the original supernatant (non-concentrated).