Opi mem medium

To assess NLRP3 inflammasome activation THP-1 cells were treated with PMA 25 ng/ml for 3 h and the cells washed once with Opi-MEM medium (Life Technologies). The cells were reseeded with 0.5 ml Opi-MEM medium in 12 well plate. LPS (50 ng/ml) was used to prime the cells overnight, and nigericin (15 µM) was added for 45 min, then the cell supernatants were collected. Bcl-2 or its siRNA were transfected into PMA treated THP-1 cells overnight. Following the culture period, the supernatants were transferred to a microcentrifuge tube and 0.5 ml of methanol and 0.125 ml chloroform added. After mixing and a 5-min centrifugation at 13,000 RPM, the upper phase was discarded being careful not to disturb the interface. 0.5 ml methanol was added the samples spun again for 5 min at 13,000 rpm. The supernatants were discarded, and the pelleted proteins air dried for 5 min at 50 °C. After which 60 µl of 1× sample loading buffer with DTT (final concentration of 0.1 µM) was added to each sample prior to SDS-PAGE and immunoblotting to detect IL-1β and caspase-1 p20.

To assess NLRP3 inflammasome activation THP-1 cells were treated with PMA 25 ng/ml for 3 h and the cells washed once with Opi-MEM medium (Life Technologies). The cells were reseeded with 0.5ml Opi-MEM medium in 12 well plate. LPS (50 ng/ml) was used to prime the cells overnight, and ATP (5 mM) was added for 1 h the following day after which the cell supernatants were collected. To assess AIM2 inflammasome activation PMA treated and LPS primed (2 h) THP-1 cells were transfected with 2 μg of Poly(dA-dT) and cell supernatants collected 6 h later. Following the culture period the supernatants were transferred to a microcentrifuge tube and 0.5 ml of methanol and 0.125 ml chloroform added. After vortexing and a 5 minute spin at 13,000 rpm the upper phase was discarded being careful not to disturb the interface. 0.5 ml methanol was added the samples spun again for 5 minutes at 13,000 rpm. The supernatants were discarded, and the pelleted proteins air dried for 5 minutes at 50°C. After which 60 μl of 1x sample loading buffer with DTT (final concentration of 0.1 M) was added to each sample prior to SDS-PAGE and immunoblotting to detect IL-1β and caspase-1 p20.