Bone marrow-derived macrophages (BMDMs) were prepared as described previously24 (link). In brief, bone marrow cells were grown in L-cell-conditioned IMDM medium supplemented with 10% FBS, 1% non-essential amino acid and 1% penicillin-streptomycin for 5 days to differentiate into macrophages. On day 5 BMDMs were seeded in 6-well cell culture plates. On the next day BMDMs were stimulated with LPS (Invivogen- LPS-SM) (20 ng/ml) or PAM3CSK4 (1 μg/ml) for the indicated hours of time followed by 5 mM ATP or 20 μM nigericin for the last 30 minutes. In some experiments, BMDMs were pretreated for 30 minutes with mouse recombinant IL-10 (50 ng/mL- Peprotech) or anti-mouse IL-10R mAb (1 μg/mL- BioxCell) before LPS/PAM3CSK4 stimulation.
In supernatant transfer experiments, supernatants from control (0 h sups), 4 h LPS treated (4 h sups) and 24 h LPS treated (24 h sups) BMDMs were transferred to fresh unstimulated BMDMs. These BMDMs were then stimulated with 20 ng/ml LPS for 4 h followed by ATP for the last 30 minutes. Caspase-1 activation was determined in the cell lysates and IL-1β and IL-18 production were determined in the supernatants by ELISA.
In supernatant transfer experiments, supernatants from control (0 h sups), 4 h LPS treated (4 h sups) and 24 h LPS treated (24 h sups) BMDMs were transferred to fresh unstimulated BMDMs. These BMDMs were then stimulated with 20 ng/ml LPS for 4 h followed by ATP for the last 30 minutes. Caspase-1 activation was determined in the cell lysates and IL-1β and IL-18 production were determined in the supernatants by ELISA.